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PLANT ANATOMY 



STEVENS 



I 



PLANT ANATOMY 

FROM THE 

STANDPOINT OF THE DEVELOPMENT 
AND FUNCTIONS OF THE TISSUES 

AND 

HANDBOOK OF MICRO-TECHNIC 



BY 



WILLIAM CHASE STEVENS 

PROFESSOR OF BOTANY IX THE UNIVERSITY OF KANSAS 



WITH 136 ILLUSTRATIONS 



PHILADELPHIA 

P. BLAKISTON'S SOX & CO 

TOI2 WALNUT STREET 
I907 



LIBRARY of CONGRESS 
Two Cooles Received 
OCT 26 •90f 
^ CopynaW Entry 

CLfSS/^ XXc, NO. 
COPY b. 



Copyright, 1907, by P. Blakiston's Son & Co. 



Press of 

The New Era Printing Company 

Lancaster, Pa. 



PREFACE. 

To one interested in biology the study of plant anatomy 
affords a rich and alluring field, since it reveals how plants, 
under conditions the most exacting, have met and solved the 
problems of their existence by achieving the power and habit 
of cell differentiation and cell association into tissues adapted 
to carry on the different physiological functions ; and when the 
study of plant anatomy is directed to reveal the process of cell 
differentiation and the steps by which the mature tissues are 
made fit for their functions, the student cannot fail to see at 
once its high biological significance. 

The ontogeny and physiology of the tissues is in fact so 
illuminating to their mature form and structure that the stu- 
dent of anatomy works at a distinct disadvantage if he is not 
constantly reverting for enlightenment to questions of origin 
and function; and whatever motive may incite him to the 
study of plant anatomy, whether it be purely intellectual curi- 
osity, or the recognition of the necessity of a knowledge of 
plant anatomy to the scientific pursuit of pharmacognosy or 
agriculture, he will find the outcome more worthy of his 
efforts if he has sought out the physiological and ecological 
interpretation of his anatomical findings. It is not nature's 
way to evolve cells and tissues at random, with no problems 
to be solved by their evolution. The tissues are not an aim- 
less expression of the power of variability. Rather, they 
represent the means of the triumph of living organisms over 
the conditions and forces which make up their environment. 

This book attempts to point out in a brief and elementary 
way how plants arrive at this achievement by the evolution of 
the different physiological tissue systems from a primitive 
undifferentiated embryonic tissue, and how the tissue systems 
are adapted by their character and relation to each other to 



VI PREFACE. 

carry out the plant's vegetative functions. It seeks to answer 
in some measure questions about what kind of organisms 
plants are; how they wrest their living from the inorganic 
world ; and how they are equipped to make satisfactory terms 
with their environment. 

At the close of each chapter are given directions for obser- 
vations that will afford a good foundation for critical discus- 
sion. In carrying out the work as there outlined the student 
will become familiar with the most important practices in 
microtechnic, and he will at the same time get training in 
independent work that will prove a significant part in his edu- 
cation. Chapters dealing in sufficient detail with microtechnic 
and microchemistry are given to help the student to pursue 
the subject beyond the limits of this book, and to undertake 
practical work in pharmacognosy and pure food and drug 
investigations. 

In the illustrations much use has been made throughout the 
book of generalized diagrams. I have found these very help- 
ful in my teaching, and I offer them here in the hope that they 
may prove suggestive to students in correlating and interpret- 
ing the details of the isolated sections with which the histolo- 
gist has to deal, and of service in throwing light on the opera- 
tion of the physiological functions. 

I must make acknowledgment of especial indebtedness for 
substance and point of view to Strasburger's Botanisches 
Practicum, and Leitungsbahnen, Haberlandt's Physiologische 
Pflanzenanatomie, Pfeffer's Physiology of Plants, Zimmer- 
mann's Microtechnic, Czapek's Biochemie der Pflanzen, Mey- 
er's Grundlagen und Methoden fur die Mikroscopische Unter- 
suchung von Pflanzenpulvern, Chamberlain's Methods in Plant 
Histology, and Winton's Microscopy of Vegetable Foods. 
The Bausch and Lomb Optical Company have kindly supplied 
the cuts for Figs. 127, 133, 135, and 136; and the Spencer 
Lens Company the cut for Fig. 131. 

I am indebted to Miss Eugenie Sterling for preparing the 
drawings for most of the illustrations, and to Mrs. Marguerite 



PREFACE. Vii 

Wise Sutton for some of the drawings. Mr. Alban Stewart 
prepared the camera lucida drawings of tissues of Avicennia, 
Psidum, and mangrove, from materials collected by him in 
the Galapagos Islands. Mr. L. M. Peace made the photo- 
micrographs appearing here, prepared many of the sections 
from which the drawings were made, and gave me many valu- 
able suggestions for the chapters on microtechnic. 

My thanks are due my colleagues, Dr. M. A. Barber, Dr. 
C. E. McClung, Dr. F. H. Billings, and Prof. Chas. M. Ster- 
ling for reading and criticizing different parts of the book, 
and to Ada Pugh Stevens for reading all of the proof sheets. 

W. C. Stevens. 
University of Kansas, 
October, 1907. 



CONTENTS 

CHAPTER I 
The Plant Cell 
The Protoplast. — Plasma Membrane or Ectoplasm. — 
General Cytoplasm. — The Nucleus.— The Plastids. — 
Cell Division. — Cell Differentiation. — Sizes of Cells. 
— The Cell Wall. — The Chemical and Physical Na- 
ture and Physiological Powers of the Protoplast. — 
Illustrative Studies 1-24 

CHAPTER II 
Differentiation of the Tissues 
General Survey. — The Primordial Meristem. — The Pro- 
toderm. — The Procambium. — The Ground Meristem. 
— The Primary Permanent Tissues. — Illustrative 
Studies 2 5~47 

CHAPTER III 
Secondary Increase in Thickness 
Dicotyledons and Gymnosperms. — Growth of the Vascu- 
lar Bundles. — Increase in the Cortex. — Monocoty- 
ledons. — Unusual Growth in Thickness. — Illustrative 
Studies 48-63 

CHAPTER IV 
Protection from Injuries and Loss of Water 
The Epidermis. — The Epidermis as a Protective Tissue. 
— The Epidermis as a Waterproof Covering. — The 
Radial and Inner Walls of the Epidermis. — The Cell 
Contents of the Epidermis. — Outgrowths and Ex- 
cretions of the Epidermis. — The Multiple Epidermis. 
— The Cork. — Cork as a Protective Tissue. — Cork 



X CONTENTS. 

as a Waterproof Covering. — Use of Cork in Healing 
Wounds. — Other Means of Protection. — Illustrative 
Studies 64-77 

CHAPTER V 
The Plant Skeleton 
The Making of the Skeleton. — The Tissues of the Skele- 
ton. — The Collenchyma. — The Bast Fibers. — The 
Wood Fibers. — The Stone Cells. — Topography of the 
Skeleton. — Illustrative Studies 7&~9 2 

CHAPTER VI 
The Absorption of Water and Minerals 
Roots in the Soil. — The Root Hairs. — Method of Intake 
of Water and Solutes. — Effect of Temperature of 
Soil, and Character and Amount of Solutes upon 
Absorption. — Absorption of Water and Solutes by 
Aerophytes. — Illustrative Studies . . 93 -][ 03 

CHAPTER VII 
Circulation of Water and Soil Solutes 
The Need of a Circulatory System. — Tissues Devoted 
to the Conduction of Water. — The Tracheal Tubes. — 
Course of Tracheal Tubes through the Stem. — The 
Tracheids. — Relation of the Tracheal Tissues to the 
Medullary Rays and Wood Parenchyma. — The Ring 
of Annual Growth. — Relation of Rings of Growth to 
Growth in Length. — Relation of Annual Rings to 
the Leaves. — Distribution of Water and Solutes 
throughout the Leaf. — The Power Concerned in the 
Ascent of Water. — Path of Water Ascent. — Influence 
of Environment on the Water Conducting Tissues. 
— Illustrative Studies 104-125 

CHAPTER VIII 
Intake and Circulation of Gases 
Oxygen and Carbon Dioxide Necessary to Plants. — The 
Stomata. — The Relation of Stomata to the Environ- 



CONTENTS. XI 

ment. — The Lenticels. — The Intercellular Spaces. — 
Diosmosis of Gases into and from Living Cells. — 
Motive Power in the Distribution of Gases through- 
out Plants. — Illustrative Studies 126-140 

CHAPTER IX 
Construction of the Plant's Food 
The Source and Uses of Food. — Food Building Appa- 
ratus. — The Chloroplasts. — The Palisade Cell the 
Chief Photosynthetic Unit. — Relation of Leaf as a 
Whole to Photosynthesis. — Conditions Affecting 
Photosynthesis. — Photosynthesis in the Lower Plants. 
— Synthesis of Food without Light. — Illustrative 
Studies 141-159 

CHAPTER X 
Circulation of Foods throughout the Plants 
Need of Circulatory Tissues. — Evidence that the Phloem 
Carries the Food. — Evidence that the Tracheal Tis- 
sues Assist the Phloem in the Upward Transmission 
of Food. — Relation of Phloem Elements to Other 
Tissues. — The Course of Food Distribution. — Annual 
Additions to the Food Conducting Tissues. — Relation 
of One Year's Phloem Elements to those of the Next. 
— Character of the Circulating Food. — The Propel- 
ling Power in Food Circulation. — Illustrative 
Studies 160-177 

CHAPTER XI 
Storage of Food and Water 
Need of Food Storage. — The Kinds of Stored Food. — 
The Process of Storage. — Location and Extent of 
Food Storage Tissues. — Fluctuations in the Solu- 
bility and Insolubility of Stored Food. — Digestion of 
Stored Food. — Assimilation of Food. — Relation of 
Stored Food to Energy Supply. — The Storage of 
Water. — Characteristics of Water Storage Tissues. 
— Illustrative Studies 178-202 



Xll CONTENTS. 

CHAPTER XII 
Secretion and Excretion 
Nature of Secretions and Excretions. — Secreting Cells 
and Glands in General. — Laticiferous Vessels or 
Milk Tubes. — Tannin Cells. — Special Enzyme-secret- 
ing Cells. — Secretion and Excretion of Minerals. — 
The Process of Secretion. — The Excretion of Liquid 
Water. — Illustrative Studies 203-217 

CHAPTER XIII 
The Preparation of Sections 
Cutting Sections Free-hand. — Cutting Sections with a 
Microtome. — Care of the Section Knife. — Cytolog- 
ical Methods. — The Fixing Process. — The Harden- 
ing Process. — The Process of Imbedding in Paraffin. 
— Sectioning Material Imbedded in Paraffin. — 
Mounting Paraffin Sections. — Staining the Sections. 
— Imbedding in Celloidin. — Staining Celloidin Sec- 
tions. — Making Permanent Mounts in Glycerine or 
Glycerine Jelly 218-237 

CHAPTER XIV 
The Use of the Microscope 
Adjusting the Microscope. — Drawing to Scale from the 
Microscope. — Use of the Polariscope. — The Use of 
Reagents on Microscopic Preparations 238-251 

CHAPTER XV 
Reagents and Processes 252-297 

CHAPTER XVI 

MlCROCHEMISTRY OF PLANT PRODUCTS 298-336 

CHAPTER XVII 
Detection of Adulterations in Foods and Drugs .... 337-343 
Index 345-349 



PLANT ANATOMY 



CHAPTER I 



THE PLANT CELL 



The plant body is composed of structural units termed cells. 
These are minute boxes often barely visible and usually en- 
tirely indistinguishable to the naked eye. The walls of the 
boxes are composed of cellulose, wood, or cork, or a substance 
allied to cork called cutin. As a rule the walls are without 
apparent perforations. The term cell has also come to include 
the living body which is always present in young cells and 
very frequently in old ones. 

The discovery that the plant body is composed of the cell 
units was made by Robert Hooke, who, about 1660, with a 
compound microscope improved by himself, saw the cellular 
structure of cork. The cells of cork have the appearance of 
the cells of honey comb, and this similarity led to his use of 
the word " cell " for the structural units of the cork, and ulti- 
mately to the extension of the usage to plants in general. The 
term is not an unfortunate one for cells considered merely as 
boxes; but its application to the living parts inclosed within 
the cell walls, or to the living part where no wall is present is 
inapt. The term protoplast (Gr. protos, first, and plastos, 
formed, the thing first formed) is now in general use to desig- 
nate the living part of the cell as a morphological unit; and 
the term protoplasm or plasma is applied to the substance com- 
posing the protoplast, just as a brick would be a morphological 
unit of a brick house, and burnt clay the substance composing 
it. Following the general usage the word cell will here be 
applied to the box and its living content taken collectively, or 



2 THE PLANT CELL 

to the box alone when the living parts have disappeared, and 
the word protoplast will be used to designate the living part 
alone. 

The box or cell wall is manufactured by the protoplast for 
its own stability and protection, and the protoplast must, 
therefore, exist before the wall which encloses it. Since the 
protoplast is the living structural and physiological unit of the 
plant body, and since everything that the plant performs is 
really the work of its individual protoplasts, it necessarily fol- 
lows that a satisfactory comprehension of plant anatomy and 
physiology is impossible without a knowledge of the nature 
of the protoplast itself. 

The Protoplast. — If we study under high magnification 
sections of an onion root tip that has been fixed, imbedded, sec- 
tioned, and put up in permanent, stained mounts as directed in 
the chapter on The Preparation of Sections, we shall find near 
the root apex young cells that have not yet secreted their cell 
walls. Each cell in this instance is therefore a protoplast and 
nothing more. It often happens in the preparation of the 
sections that some of the protoplasts become separated from 
the others and so reveal to us just how much constitutes a 
single protoplast. Fig. I, A, shows us such a protoplast. In 
the root all of the parts here shown were living, excepting 
possibly the nucleolus b. The cytoplasm c constitutes the bulk 
of the protoplast ; the nucleus a is imbedded in the cytoplasm ; 
the plasma membrane d is a specialized outer portion of the 
cytoplasm; the plastids e are relatively very minute parts of 
the protoplast, but have a special work to do, as will be learned 
later on. In older cells farther back from the root tip we 
find that the protoplasts have secreted a wall, as shown in 
Fig. i, B. 

Comparing A and B of Fig. i we see that in the younger 
protoplast the cytoplasm looks something like a sponge with 
very fine meshes while in the older protoplast the cytoplasm 
does not so completely fill out the space inclosed by the plas- 
matic membrane, some of the meshes having widened into 



PLASMA MEMBRANE 3 

relatively large rifts. It seems that as the protoplast grows 
older the cytoplasm does not keep pace with the general in- 
crease in size and its network in consequence becomes broken 
in places, allowing the small meshes to coalesce into larger 
ones known as vacuoles. Both the meshes and the vacuoles 
are filled with cell sap. The cell sap is largely water contain- 
ing in solution salts that have come up from the soil, and food 
substances together with various other compounds manu- 
factured within the plant. 

After this general view of the several parts of the proto- 
plast we are ready to examine into the character and uses of 
each more thoroughly. In doing this let us not lose sight of 
the fact that the protoplasts taken collectively constitute the. 
living body of the plant and whatever the plant 'does as a liv- 
ing organism is accomplished by them. They put to use 
various forces of the external world; they compound the 
plant's food and a multitude of other products, many of 
which are employed by the pharmacist; they make the cell- 
wall framework of the plant; they multiply and increase in 
size so that the plant as a whole is made to grow ; they differ- 
entiate the various tissues, each suited to perform a particular 
service; they are sentient to gravity and light, moisture and 
temperature, and to the general state of the whole organism 
of which each protoplast forms a part, and they are capable 
of responding to these things in a definite and useful way. 
With this knowledge we might anticipate, and it is not sur- 
prising to find, that each protoplast is a complex thing with 
visibly distinct parts, and that each part has its own physio- 
logical significance. We shall now take up the parts of the 
protoplast in the following order: plasma membrane, general 
cytoplasm, nucleus, plastids. 

Plasma Membrane or Ectoplasm. — At the exterior of 
every protoplast is a very thin, hyaline membrane, which, as 
has been said, is morphologically a part of the cytoplasm, for 
when a protoplast is torn or cut in two a membrane is pro- 
duced from the cytoplasm over the wounded surface. This 
membrane is known as the plasma membrane or ectoplasm 



THE PLANT CELL 



(Fig", i, A, a). It is approximately .0003 mm. thick, or about 
1/400 of the thickness of this page. After the cell wall has 

been formed it is very diffi- 
cult to distinguish the plas- 
ma membrane because of its 
extreme thinness and close 
contact with the wall; but 
the greater part of this diffi- 
culty vanishes when the 
protoplast is made to shrink 
away from the wall. This 
we can do to advantage with 
the epidermal cells of Tra- 
descantia zebrina containing 
a colored cell-sap. We strip 
a bit of epidermis from the 
under side of a midrib of 
a leaf and mount it under 
a coverglass in a drop of 
water. We bring some of 
the colored cells under the 
objective and run a 5 per 
cent. NaCl solution under the 
coverglass. The salt solu- 
tion draws water out of 
the cells by osmosis and the 
protoplast soon shrinks 
away from the walls because 
it is elastic and had been 
stretched by the water within 
the cell. We can now 
make out the thin plasma 
membrane at the surface of 
the shrunken protoplast (Fig. 1, C). If we replace the salt 
solution under the coverglass with fresh water the protoplast 
quickly swells up and presses against the cell wall all around 
as before. 




Fig. 
root-tip ; 
plasm; 



t. A, embryonic cells from onion 
d, plasmatic membrane; c, cyto- 
, nuclear membrane enclosing the 
threadlike nuclear reticulum; b, nucleolus; 

e, plastids (black dots scattered about). 
B, older cells farther back from the root- 
tip. The cytoplasm is becoming vacuolate; 

f, vacuole. C, a cell from the epidermis 
of the midrib of Tradescantia zebrina, in 
its natural condition on the right, and 
plasmolyzed by a salt solution on the left; 

g, space left by the recedence of the cyto- 
plasm from the wall; the plasma membrane 
can now be seen as a delicate membrane 
bounding the shrunken protoplast. All highly 
magnified. 



GENERAL CYTOPLASM 5 

In this experiment we can see that while the plasma mem- 
brane allows the water to be drawn from the protoplast by the 
salt solution it does not permit the coloring matter or the 
osmotic substances in solution in the cell sap to escape, for the 
color does not at all diminish in the protoplast as it shrinks, 
and the protoplast could not again swell up on the replacement 
of the salt solution by water if its osmotic substances had been 
lost. This guardianship of the exchange of materials between 
the cells or between the cells and the external world is one of 
the well-recognized functions of the plasma membrane. Since 
the plasma membrane lies at the surface of the protoplast it 
must receive and transmit to the other parts the stimuli that 
come from without. Until the cell wall is built the plasma 
membrane doubtless affords some rigidity and protection to 
the parts within; and when the time for the building of the 
cell wall arrives it seems that the plasma membrane constructs 
it by the chemical transformation of its own substance into 
the substance of the wall. 

General Cytoplasm. — The cytoplasm is the living matrix 
in which the nucleus and plastids are imbedded. In very 
young cells it fills out all of the space not occupied by the 
nucleus and plastids (Fig. i, A), but in old cells it becomes 
a very thin film, hardly greater than .0006 mm. in thickness 
lining the cell wall. In cells that have been killed, fixed and 
stained in the usual ways (see Chapter on The Preparation 
of Sections) the cytoplasm has a spongy or netted appearance 
(Fig.l,A). 

It is uncertain whether the cytoplasm is really spongelike 
with irregular and intercommunicating canals, or alveolar with 
each cavity a closed sac. Whatever the exact character of 
the cavities may be they are filled with cell sap, or, in many 
instances, with insoluble reserve food, such as starch, proteids, 
and oils, and excretions, such as crystals of calcium oxalate. 

As has been stated, as the cell grows older some of the cavi- 
ties in the cytoplasm enlarge and coalesce, and are then known 
as vacuoles. A plasma membrane is formed about the vacu- 



O THE PLANT CELL 

oles similar to the exterior plasma membrane already described, 
and it exercises a selective function over the passage of mate- 
rials to and from the vacuole just as does the exterior mem- 
brane to and from the protoplast as a whole. The spongy, 
or alveolar, condition of the cytoplasm persists until the divi- 
sion of the nucleus preparatory to cell division sets in, when a 
part becomes threadlike and seems to assist in the translocation 
of the chromosomes (the definite parts into which the nuclear 
substance becomes segmented during nuclear and cell division) 
to the opposite poles of the cell; this process may, therefore, 
be classed as one of the functions of the cytoplasm (see Fig. 3). 

Throughout the life of the cell the cytoplasm has many 
things to do of a chemical nature, but it is very improbable 
that it often works independently of the nucleus. Where 
storage of proteids and oils is taking place we find them stowed 
away in the meshes of the cytoplasm, and this is good circum- 
stantial evidence that the cytoplasm has manufactured them 
where we find them. This is particularly true of the insoluble 
proteids. The cytoplasm probably secretes the ferments by 
means of which the stored materials are digested when they 
are wanted for food. Many other of the ceaseless activities 
of the cell are doubtless accomplished with its assistance. 

The Nucleus. — In young cells the nucleus is spherical in 
form and lies at the center imbedded in the cytoplasm and 
occupies from .5 to .8 the diameter of the cell. It consists 
of the nuclear membrane, nuclear reticulum, and nuclear sap, 
and usually contains one or more nucleoli (Fig. 1, A, b). The 
nuclear membrane appears to be really a part of the cytoplasm, 
similar to the plasmatic membranes lining the exterior of the 
cell and of the vacuoles. The reticulum is the essential living 
part of the nucleus. The nuclear sap appears to be a fluid 
which furnishes to the reticulum water and food, and in other 
ways serves it; while the nucleolus seems to be reserve food 
of a peculiar kind needed to help in the processes of nuclear 
and cell division. 

The nucleus is often spoken of as the center of life of the 



THE NUCLEUS 7 

cell. While the statement is vague it conveys a meaning not 
entirely misleading-. Some of the facts at the foundation of 
this conception are these: (a) When a protoplast is segmented 
into two parts by plasmolysis or other artificial means the part 
containing the nucleus has the power to construct a wall about 
itself, while the enucleated part has not. Although the plas- 
matic membrane is the immediate agent in the construction of 
the wall it can not do its work without the influence of the 
nucleus, (b) We have good reasons for the belief that the 
nucleus is the cause of oxidation in the living cell, and, without 
it, the tearing down and building up processes of the cell that 
depend upon oxidations can not go on. (c) After the nucleus 
has been removed the remainder of the protoplast soon dies. 
While, on these grounds, the nucleus may be spoken of as the 
center of life of the cell, the fact must not be left obscured 
that a nucleus dissociated from the rest of the protoplast can 
not long maintain its existence, and the cooperation of the 
other parts is necessary to the success of the functions of the 
nucleus. 

The nucleus is also frequently spoken of as the bearer of 
the inheritable characters and qualities. This, too, is not 
without foundation, and if true the nucleus stands forth as 
the architect and master of the form, character, and activi- 
ties of the cell and entire plant body. The grounds for the 
assumption are: (a) In the embryonic cells of the growing 
apex or cambium the nucleus is relatively large, having an 
average diameter 0.6 that of the entire cell. These cells are 
to undergo profound changes in size, form, and general char- 
acter as the differentiation of tissues proceeds, and why should 
the nucleus be allotted so much space within the cells unless 
it plays a dominant part in the hereditary differentiations that 
are to follow ? The evidence here is enough to raise the ques- 
tion, but it does not go far towards solving it. (b) During 
nuclear and cell division the nuclear reticulum, which is the 
essential part of the nucleus, divides into many pieces with 
great precision, and these pieces are equally distributed between 



8 THE PLANT CELL 

the nuclei of the two resulting cells. What but the vehicle 
of hereditary transmission should require such exactness of 
distribution? Here again the facts afford us hardly more 
than the suggestion of the question, (c) In fertilization, 
when the sperm cell from the pollen grain is fusing with the 
egg cell within the ovule, the sperm cell consists almost entirely 
of nucleus, and the egg cell is relatively rich in cytoplasm ; yet 
the offspring may partake as much of the peculiarities of the 
male as of the female parent. The inference is that, since 
the male contributed little more than a nucleus and the female 
a relatively large amount of cytoplasm in addition to the 
nucleus, it must be that to the nucleus have been given the 
inheritable qualities to transmit from generation to genera- 
tion; for if the cytoplasm bears these qualities equally with the 
nucleus the impress of the female might be expected to pre- 
ponderate in the offspring, {d) In the nuclear and cell divi- 
sions resulting in the production of pollen spores and embryosac 
spores the number of chromosomes (see Fig. 3) in each 
nucleus is reduced one half. The sperm cell and egg cell 
arising from the germination of pollen spore and embryosac 
spore each contain the reduced number of chromosomes, but 
when they fuse in the act of fertilization the original number 
is restored in the fertilized egg and so continues throughout 
the body of the resulting offspring until pollen spores and 
embryosac spores are again produced by it with the reduced 
number. What the significance of this halving and doubling 
may be is still open to debate, but the meaning evidently will 
be found in relation to the transmission and blending of the 
male and female qualities. Since the chromosomes are seg- 
ments of the nuclear reticulum the phenomenon here described 
may be classed as a part of the evidence that the nucleus is the 
bearer of the inheritable qualities, (e) A new line of evidence 
is at present being opened up in the study of hybrids, where 
the chromosomes from each parent are found to retain their 
individuality in the offspring. This fact is being used to illu- 
minate some problems in heredity, and if future investigations 



THE PLASTIDS 9 

confirm the conclusions that are being drawn from present 
evidence the hereditary function of the nucleus will be firmly 
established. 

The Plastids. — The plastids are distinct and usually rela- 
tively small parts of the protoplast. They vary in size from 
barely discernible points with the highest magnifications to 
discs and bands that traverse the entire length of the cell. In 
form they may be orbicular, ellipsoidal, disc, or ribband shaped. 
They are classed under three names according to their color: 
the leucoplasts are colorless ; the chloroplasts contain the green 
coloring matter chlorophyll; the chromoplasts are yellow, 
orange, or red. They are, however, really the same thing 
under different guises and performing different functions; 
for the leucoplasts may become chloroplasts, and the chloro- 
plasts chromoplasts. This fact is well made out in the tomato, 
for instance, where the very young pistil in the bud contains 
no other plastids than the leucoplasts and is colorless in con- 
sequence; later when the corolla drops away and the pistil 
emerges into the light the leucoplasts produce chlorophyll and 
the young fruit is green; but when the fruit begins to ripen 
the chlorophyll is gradually replaced by red and orange color- 
ing matters, and the chloroplasts become chromoplasts. The 
leucoplasts are therefore the progenitors of the other plastids, 
but they have their own functions to perform as leucoplasts : 
they take carbohydrates out of solution in the cell sap and 
store these within themselves in the form of insoluble starch 
grains. This is well seen in those cells and tissues, as in the 
potato, and the endosperm of seeds, where reserve food is 
stored away. The chloroplasts secrete two coloring matters 
known as chlorophyll-green and carotin or chlorophyll-yellow. 
The chloroplasts employ these pigments in arresting the sun's 
energy, by means of which they make the food of the plant. 
In Chapter 9 the chloroplasts will be more fully discussed in 
connection with the photosynthetic system. The chromoplasts 
impart red, orange, and yellow colors to flowers and fruits, 
where, within the chromoplast body, the reds occur as crystal- 



10 



THE PLANT CELL 



line carotin and the yellows as amorphous xanthin. These 
may be found separately or together in the same chromoplast. 
The presence of both produces an orange color (Fig. 2). The 
reddish and bluish pigments occurring in solution in the cell 
sap are known as anthocyanin, and it does not appear that the 
chromoplasts are necessary to their production. 




Fig. 2. A, cell from the epidermis of the upper side of the calyx of Tropaeolum 
majus with crystalline chromoplasts; B, cells from the petal of Lupinus luteus with 
yellow chromoplasts; C, cell showing numerous chloroplasts scattered through the 
cytoplasm. (A, after Strasburger; B, after Frank.) 



Cell Division. — In the growing apices of root and shoot 
and in the cambium cell division may go on indefinitely. The 
process of cell division begins in the nucleus and terminates 
by the formation of a dividing cell wall, as will be seen in Fig. 
3, where the different stages may be followed. The nuclear 
reticulum (in a) becomes transformed into a thick winding 
thread (in b), which in successfully stained sections is seen 
to consist of colored discs or granules termed chromatin, im- 
bedded in a colorless matrix called linin. The thread splits 
longitudinally throughout its length (in c), and then breaks 
into rod-shaped pieces, each of which consists of two longi- 
tudinal halves arising from the longitudinal division of the 
thread (in d). These rods are known as chromosomes, and 
their number varies with the species. Next the nuclear wall 



CELL DIVISION 



I I 



disappears and threads arise in the cytoplasm and converge to 
a point at two opposite poles forming what is known as the 
nuclear spindle (in e). Some of the threads extend uninter- 
ruptedly from pole to pole while others become fastened to the 







wm 





f 


r~. 


# 




L 


in 



I 

Fig. 3. Semi-diagrammatic representation of nuclear and cell division, a, resting 
cell ready to begin division; b, the nuclear reticulum is assuming the form of a 
thickened thread, and the cytoplasm at opposite poles is becoming threadlike to form 
the spindle fibers; c, the nuclear thread has divided longitudinally through the middle, 
and the spindle fibers have become more definite; d, the nuclear membrane and the 
nucleolus have disappeared, and the nuclear thread has become segmented into chromo- 
somes which are assembling at the equator of the cell. All of the phases of division 
thus far are called prophases, e, the metaphase, where the longitudinal halves of the 
chromosomes are being drawn apart preparatory to their journey towards the opposite 
poles; f, the anaphase, or movement of the chromosomes towards the poles, is about 
completed, connecting fibers extend from pole to pole; g, telophase where the chromo- 
somes have begun to spin out in the form of a nuclear reticulum. The connecting 
fibers have begun to thicken in the equatorial plane; h, the connecting fibers have 
spread out and come into contact with the wall of the mother-cell in the equatorial 
plane, and the thickening of the fibers throughout this plane has made a complete 
cell plate within which the dividing wall will be produced; i, a nuclear membrane has 
been formed about each daughter nucleus, and the dividing cell wall is completed. 
The two daughter cells are now ready to grow to the size of the parent cell in a, when 
the daughter nuclei will appear as does the nucleus there. All highly magnified. 



12 



THE PLANT CELL 




chromosomes. The chromosomes in some unknown way line 
up in an equatorial plane half way between the poles, and then 
one half of each chromosome is drawn to one pole and the 
remaining half to the other where they form at each pole 
a nuclear thread (e, j, and g). This spins itself out into 

a nuclear reticulum around 
which a nuclear membrane is 
soon organized (in h and i). 
The connecting fibers ex- 
tending between the poles 
bulge out at the equator 
more and more and new 
ones evidently are formed 
until the whole equatorial 
zone is traversed by them 
(in g and h). The fibers 
thicken in the equatorial area 
and the thickenings fuse 
together, forming a closed 
membrane known as the cell 
plate (progressive stages in 
g, h, and %). This is a plas- 
matic membrane which organizes in its median plane a cell 
wall dividing the mother cell into two. The fibers disappear, 
the cytoplasm becomes reticulate throughout, and the daughter 
nuclei increase rapidly in size. 

About the time when the nuclear membrane disappears the 
nucleolus as a rule also vanishes from sight and can not again 
be found until the completion of the daughter nuclei. This 
has led to the conclusion that the nucleolus is not a living part 
of the protoplast but is simply a form of reserve food needed 
at the time of nuclear and cell division. It does not, however, 
behave uniformly in all subjects and its exact nature is still 
a subject of debate. 

In the nascent endosperm of seeds the division wall between 
daughter cells may not be formed until the nuclei have many 



Fig. 4. Formation of endosperm in the 
embryo-sac of Agrimonia Eupatorium. Cell 
walls are being formed between the nuclei. 
(After Strasburger.) 



CELL DIVISION l J 

times divided. Finally, when the embryosac containing the 
endosperm lias completed its growth, new connecting- fibers 
spring up in the cytoplasm between the nuclei, and cell walls 
are laid down in the usual way (Fig. 4). 

In the formation of spores in the Ascomycetes nuclear divi- 
sion takes place in the usual way, but the method of formation 
of the wall about the spores is unique. Here nuclear division 
continues within the mother cell until the number of nuclei 
equals the number of spores to be produced, when, at the close 





Fig. 5. Free-cell formation of spores in the ascus of Erysiphe communis. A, ascus 
with single nucleus; C, cytoplasm; N, nucleus; NL, nucleolus; B, successive stages in 
nuclear division within the ascus; at X, early anaphase, nuclear membrane, NM, 
still persisting; R, kinoplasmic radiations from the poles; at Y telophase, new nuclear 
membrane not yet formed; Z, a later stage where the nuclear membranes demark the 
daughter nuclei; C, A, B and C, are successively later stages than Z in B. At A 
some of the kinoplasmic radiations are bending downward about the nucleus N; at B 
the nucleus is nearly enclosed; and at C entirely enclosed by the radiations, which 
now form a complete membrane cutting off a portion of the cytoplasm of the ascus, 
and thus forming a complete cell. (Arranged after drawings by Harper. B and C 
are diagrammatic. For the sake of simplicity of description various stages of nuclear 
division are shown in a single ascus, although at any given time only one stage would 
actually be present.) 



of the last nuclear division fibrillar radiations from the pole 
end of the nuclei bend back about the nucleus and finally by 
their fusion form a complete plasma sac enclosing, together 
with the nucleus, a part of the cytoplasm of the mother cell 
(see Fig. 5). All of the spores thus produced lie uncon- 



14 



THE PLANT CELL 





Fig 
by budding 
Reess.) 



nected within the mother cell. This is known as free cell- 
formation. 

In Spirogyra and other cases among the Thallophytes the 
dividing cell wall is produced by a gradual ingrowth from the 

wall of the mother cell 
between the two daugh- 
ter nuclei which have 
previously been form- 
ed. This process of 
wall formation reminds 

6. Various stages of cell multiplication ° ne °I the gradual 
of Saccharomyces cerevisice. (After closing in f mm all sides 

of an iris diaphragm. 

In yeasts and some other fungi cell division is preceded by 
budding. The mother cell puts forth an outgrowth like itself 
in general form; the nucleus divides and one of the daughter 
nuclei enters the bud ; and a division wall is then formed sepa- 
rating the bud from the parent cell (Fig. 6). 

The kind of nuclear division above de- 
scribed is called indirect, mitotic, or karyo- 
kinetic division. Another method called 
direct or amitotic division is where the 
nucleus simply constricts itself into two 
(Fig. 7). This is of rare occurrence, being 
found chiefly in old cells that have about 
run their course. 

Cell Differentiation. — The new cells 
formed by the dividing cells of the growing 
apices and cambium do not long remain in 
size, form, and other characteristics just as 
they are when first produced. In cross-sec- 
tions of the growing point of Aristolochia, 
for example (Fig. 8), the cells are essentially 
all alike ; but a little farther down the stem we find zones and 
groups of tissues which are readily distinguished from each 




Fig. 7. Nucleus 
dividing by simple 
constriction. From 
the lining of the 
embryo-sac of Vicia 
faba. (After Zim- 
mermann.) 



CELL DIFFERENTIATION 



15 



other because the cells composing them differ in size, form, 
thickness of walls, etc. These cells and tissues have all arisen 
by changes in cells that were produced by cell division at the 
growing point. It appears that 
these cells, influenced by stim- 
uli, and impelled by impulses 
mysterious to us, set to doing 
different things; some doing 
little more than enlarging uni- 
formly; some enlarging and 
thickening and chemically 
changing their walls; others 
greatly elongating and becom- 
ing fibers and tubes. It is 
this power of the living cells 
to become different things that 
has made possible division of 
labor in the plant body, with 
the consequent successful oc- 
cupation of all sorts of habi- 
tats, and the commanding size 
of our trees and shrubs. 

Sizes of Cells. — After its for- 
mation by cell division the cell 
grows to its adult size which 
varies within not very wide 
limits in each tissue. An aver- 
age size of cells, not much elon- 
gated like wood and bast fibers, 
is approximately .03 mm., or 
about one fourth the thickness 
of one of these pages. Several 
reasons can be suggested for 
this habitually small size: (a) 
The plant is made stronger 
thereby. The cell walls must 




Fig. 8. i, cells from a cross- 
section, and 2, from a longitudinal 
section through the primordial meri- 
stem of the growing apex of Aris- 
tolochia sipho; the cells are essen- 
tially alike from both points of 
view. 3, 4 and 5 show cells from 
some of the different tissues which 
the primordial meristem produces. 
Note the different shapes and thick- 
nesses of walls. 3 is from the 
sclerenchyma ring, 4, from the col- 
lenchyma, and 5, from the epider- 
mis. All magnified to the same 
scale. 



l6 THE PLANT CELL 

be, on the whole, extremely thin to permit an easy interchange 
of materials, and the smaller the cells the stronger they will 
be with a given thickness of wall. The smaller the cells the 
greater the number of walls in a given volume of tissue, so 
that the whole tissue is made stronger. (&) Each cell is a 
chemical laboratory, each nucleus a center of oxidations, so 
that the more there are of these the greater the activity of the 
whole body, (c) The smaller the cells the greater the amount 
of -protoplastic surface exposed for the osmotic interchange 
of materials between contiguous cells and between cells and 
intercellular spaces, (d) The smaller the cells the greater the 
number of nuclei to send forth hereditary stimuli which must 
dominate every part of the body. All of these problems are 
of great moment to the well being of plants and the minute 
size of the cells is one of the factors in their solution. 

The Cell Wall. — The cell wall is the skeleton of the proto- 
plast, preserving its form and protecting it from danger; it 
also gives rigidity and strength to, and preserves the form of, 
the whole plant body. When first formed the wall is usually 
cellulose «(C 6 H 10 O 5 ) ; we know, however, that we are includ- 
ing under this name a group of closely allied substances which 
yield different products on chemical decomposition. 

The wall may remain cellulose throughout the existence of 
the plant, or it may become modified in different tissues to 
meet special requirements. Thus, in the bast and wood fibers, 
the walls become lignified ; and in the epidermis and cork they 
are cutinized and suberized. It is not known precisely what 
the chemical nature of these modifications is, but lignification 
seems to increase the hardness, strength, and elasticity, with- 
out decreasing the permeability of the walls; while cutiniza- 
tion and suberization involve infiltration with waxes, so that 
the walls are made more or less impermeable to water and 
gases, as happens to paper when infiltrated with melted 
paraffin. Sometimes the wall undergoes a mucilaginous modi- 
fication by which, if the modification is extensive, it swells 



NATURE AND POWERS OF PROTOPLAST 1/ 

enormously on coming in contact with water. Walls of this 
kind occur at the surface of many seeds, such as flax and 
mustard, where they are useful in gluing the seeds to the sub- 
stratum ; and they are frequently found in desert plants where 
they are useful in imbibing and holding stores of water. 

The cell wall when first formed is relatively very thin, and 
its growth in thickness and extent is accomplished by the 
addition of new particles within it (growth by intussusception) 
and at its surface (growth by apposition). On account of the 
unequal swelling of the cell walls in water in its different 
dimensions, and its behavior like a crystal in polarized light, 
Xageli has conceived the hypothesis that it is composed of 
aggregations in crystalline form of minute parts or molecules 
to which he has given the name micellae. He conceives that 
the micellae are separated by films of water which become 
thicker on the swelling and thinner on the drying up of the 
wall. This hypothesis still explains better than any other the 
optical properties of the cell wall and the phenomena of 
imbibition. 

The Chemical and Physical Nature and Physiological 
Powers of the Protoplast. — The exact chemical constitution 
of the protoplast is not known. The evidence thus far at 
hand goes to show that it is chiefly a complex of proteids of 
very large molecules where the elements carbon, hydrogen, 
oxygen, and nitrogen are always present, and frequently sul- 
phur, and, in the nucleus, phosphorus. Water is always asso- 
ciated with the protoplast and is necessary to its life. Other 
substances than the proteids and water are sometimes, and 
may be always, present. On account of its complex nature the 
plasma or substance of the protoplast is easily broken down 
into simpler substances, setting free large amounts of energy; 
and correspondingly large amounts are required for its growth 
and repair. 

In consistency the protoplast is apparently semi-fluid. We 
get this conception from the rotation and circulation of the 
cytoplasm in Nitella, stamen hairs of Tradescantia, Myxomy- 



10 THE PLANT CELL 

cete plasmodia, etc. Here the cytoplasm simulates a fluid in 
its streaming movements, while it preserves its form sharply 
demarked from the surrounding cell sap. The plasmodium of 
Myxomycetes consists of a mass of cytoplasm with many im- 
bedded nuclei, and no cell walls are present; so that its con- 
sistency, which is about that of thin batter, may be taken as an 
indication of the consistency of protoplasts in general. The 




Fig. 9. 1 and 2, nuclei from the tapetal plasmodium of the sporangium of 
Botrychium Virginianum, 1, in the usual form, and 2, flattened while entering a 
crevice between spore-groups; 3, cell from Nitella showing rotation of the cytoplasm 
as indicated by the arrows; 4, cell from stamen-hair of Tradescantia, showing circula- 
tion of the cytoplasm as indicated by the arrows. 



nucleus and plastids seem to be firmer and less fluid than the 
general cytoplasm, but their mobility is shown by the apparent 
ease with which they change their form ; as when chloroplasts 
round themselves up or flatten out with varying intensities of 
light, and nuclei of tapetal cells flatten themselves and creep 
into very narrow rifts between groups of developing spores 

(Fig. 9). 

The things which the protoplast is capable of doing as a 
physiological unit have partly been told in the foregoing and 
will be further brought out in succeeding chapters. By way 
of convenient oversight these activities will now be grouped 
and classified : 

(a) Absorption. — Water, and solids and gases in solution, 



NATURE AND POWERS OF PROTOPLAST I (J 

are drawn through the plasma membranes into the meshes of 
the cytoplasm, vacuoles, and nuclear cavity. The powers of 
osmosis and diffusion are at work here, essentially as we find 
them in artificial cells and through artificial and lifeless mem- 
branes; but the chemical work done by the living protoplast 
makes and keeps up the conditions necessary to osmosis and 
diffusion, and the plasma membranes modify, and to a certain 
extent regulate, the osmotic and diffusion exchange of materi- 
als into and from the body of the protoplast. 

(b) Construction. — The protoplast builds complex sub- 
stances from simpler ones, as when sugar, starch, oil, and pro- 
teid foods are built from carbon dioxide, water, and salts of 
nitrogen and sulphur; and when the foods are assimilated to 
the substance of the protoplast itself. 

(c) Destruction. — In respiration the protoplast breaks down 
a part of its own substance, and probably also reserve foods, 
into simpler compounds ; and the self-destruction of the proto- 
plast appears to take place in the production of secretions, such 
as cell wall and digestive ferments. 

(d) Excretion. — Frequently substances that are of no use 
are excluded from the protoplast by inclosure in an insoluble 
form in vacuoles, or deposited in the cell walls, or thrown into 
intercellular spaces ; or useful substances, such as enzymes, and 
acids for making intractable substances soluble, and nectar for 
alluring insects, may be expelled at the surface. This isola- 
tion by the protoplast of substances from the sphere of its 
operations is called excretion. 

(e) Contraction and Expansion. — When the protoplasts are 
not hemmed in by a cell wall, as in Euglena, the plasmodium of 
Myxomycetes, and zoospores (Fig. 10), they are found to be 
capable of alternate contraction and expansion, resulting in 
marked change of form in Euglena and plasmodia, and in the 
thrashing back and forth of the cilia of swarm spores; and the 
circulation and rotation of the cytoplasm spoken of above is 
probably caused by contraction and expansion in its different 



20 



THE PLANT CELL 



parts. The power of contraction and expansion is usually 
comprehended in the term contractility. 

(/) Perception and Response. — The protoplast is capable of 
perceiving external stimuli and responding to them in a defi- 
nite way. Thus, when the 
Light swarm-spores of Ulothrix are 

mounted in a drop of water 
for examination with the micro- 
scope they are found to swim 
to the side of the drop next the 
window, but if direct sunlight 
is allowed to fall upon them 
they recede to the opposite side. 
It is clear that they perceive 
the light and behave as they do 
on account of its varying inten- 
sity; and it appears that they 
not only perceive the light, but 
also the direction from which 
it is coming. The inclining of 
house plants towards the win- 
dow is another example of this 
kind. When certain kinds of 
bacteria are mounted in a drop 
of water under a cover glass they 
are found to assemble about 
the edge of the coverglass to get oxygen; in which case the 
stimulus is of a chemical nature; and in fertilization the 
finding of the egg by the sperm cell seems to be due to the 
perception of a chemical substance excreted by the egg. The 
growth of roots towards the center of the earth and of shoots 
away from it is due to a perception of the direction of the 
gravity pull. Many other instances could be given for stimuli 
of different natures; but the few here cited will do to show 
the fact of perception and response by the protoplasts. This 
power of the protoplasts is called irritability. 




Fig. io. i, a bit of Plasmodium 
of a Myxomycete, arrows showing di- 
rection of movement of the cytoplasm; 
2, Euglena viridis in different stages of 
contractility; 3, ciliated swarm-spores 
of Ulothrix zonata; 4, diagram indi- 
cating movement of swarm-spores of 
Ulothrix towards the source of light. 



NATURE AND POWERS OF PROTOPLAST 2 1 

(g) Growth. — When protoplasts are permanently increas- 
ing in size and assuming their permanent forms they are grow- 
ing. Examples of this are seen where cells at a growing apex 
or in the cambium region are increasing in size and taking on 
the forms characteristic of the different tissues which they are 
to compose. Of course the size and form of the whole plant 
body are due to the growth activity of its individual protoplasts. 

(/?) Reproduction. — We have already seen that the proto- 
plast is able to reproduce its kind by self division, where the 
behavior of the nucleus indicates that the process is one of 
considerable complexity. 

Illustrative Studies 

i. Cut free-hand sections (see page 218) of bottle cork from 
the three points of view illustrated in Fig. 123, and mount 
them for study in a drop of dilute glycerine (half glycerine 
and half water). Draw three or four cells from each point 
of view. If the microscope is equipped with a micrometer 
eyepiece — and it should be for the work of this book — draw 
to scale as explained on page 245. In drawing cell groups out- 
line the cell cavities, leaving a space between adjoining cells 
to represent the thickness of the cell walls, as shown in Fig. 
i,B. 

2. Study longitudinal sections of onion root tip prepared 
as directed under Cytological Methods on page 225. Or if 
this cannot be done study cross-sections of onion root tips 
that have been prepared by fixing the tips in chrom-acetic 
fixative (page 227) and cutting the sections free-hand while 
the tips are enclosed in elder pith (page 218), and finally stain- 
ing in safranin and mounting in dilute glycerine (page 236). 

To secure the onion root tips boil a disk of carpet paper to 
moisten it and kill possible fungal parasites, lay it on glass 
or tile, place a few onions with basal ends on the moist paper, 
and cover with a bell- jar. When the roots are about 3 mm. 
long cut them off and place them in Flemming's fixative (page 
226). Proceed as directed under Cytological Methods until 



2 2 THE PLANT CELL 

the sections are stained in safranin-gentian violet-orange, or 
in iron-hsematoxylin (page 233), and mounted in balsam. 

Find all the parts of the cells as described in the chapter. 
Draw younger and older cells to scale, using Fig. 1 as a 
model for line-work and stippling. By means of the eyepiece 
micrometer determine in millimeters the diameters of the 
cells, nuclei, and cell walls. The plastids are so small here 
that they cannot be identified with certainty. If the sections 
show nuclear and cell division, as they should if the sections 
have been properly prepared, find the different stages illus- 
trated in Fig. 3 and draw them to scale. 

3. Mount a fresh moss leaf in dilute glycerine and study 
the chloroplasts with a high power. Draw to scale a single 
cell with its contained chloroplasts. Measure the chloroplasts. 
Possibly some of them will be found dividing by constriction. 
With a colored pencil tint the drawing of the chloroplasts 
green. 

4. Make free-hand sections of the ray florets of Zinnias of 
different colors while holding the florets in elder pith (page 
218). Mount the sections in dilute glycerine and study them 
quickly before the pigments that are in solution in the cell 
sap have time to escape. Find the yellow and orange chromo- 
plasts in some sections and colored cell sap in others, and 
sometimes a combination of both in the same section. Draw 
a few cells in each case and use colored pencils to express the 
colors. 

5. Crush out between coverglass and slide a very small bit 
of the pulp of a tomato or of climbing bitter-sweet berries- 
using the juice of the fruit as a mounting medium. Draw and 
measure chloroplasts of different forms and colors. Tint the 
drawings as near the natural colors as possible. 

6. To see in a comprehensive way that cell walls may be 
of different kinds mount thin cross-sections of old stem of 
Aristolochia (Fig. 24), or of some other woody stem, in dif- 
ferent reagents for differentiating cell walls. Examine a sec- 
tion first in water to note the natural colors of the walls. 



ILLUSTRATIVE STUDIES 23 

Filter away the water and add a drop of chloroiodide of zinc. 
After a while the cell walls of some of the tissues will be 
colored yellow and others purple. The purple walls are cellu- 
lose and the yellow are lignified, suberized or cutinized. 

Mount another section in aniline sulphate and only the lig- 
nified walls will be colored yellow while the others will be left 
unstained. Mount a third section in phloroglucin and the 
lignified walls will be colored pink while all others will be 
unchanged. Finally sections left for several hours in a tinc- 
ture of alcannin will have the cutinized and suberized wall 
alone stained pink. See under these reagents in Chapter XV. 

7. Mount a bit of Nitella in a drop of water and study the 
flow of the cytoplasm. With a micrometer eyepiece and a 
metronome to tick off seconds determine the rate of flow per 
second. 

Remove a filament from a young bud of Tradescantia Vir- 
ginica and mount it in a drop of water. Study the circulation 
of the cytoplasm in one of the cells of a hair growing from 
the filament. How does it differ from Nitella as to the man- 
ner and rate of flow? Draw a cell, using Fig. 9 as a pattern 
for line-work and stippling. 

8. Mount in water some of the mealy green scum some- 
times found at the surface of stagnant ponds. If it is found 
composed of motile green bodies with a red eyespot these indi- 
viduals are Euglena viridis. Note the beating back and forth 
of the colorless flagellum at the eyespot end, and show by 
drawings the different forms which a single Euglena is found 
to assume within a short time. 

9. Make a culture of yeast as follows : Pare, slice thin, and 
boil a good-sized potato in a pint of water. After boiling till 
it is soft mash it fine and add to this potato broth a table- 
spoonful of sugar. Pour a little of the broth into a test-tube 
(1) for future use and after the remainder cools dissolve in 
it a piece of yeast cake and set the culture in a warm place. 
After four or five hours when the culture is beady with bub- 
bles, indicating that the yeast is rapidly multiplying, remove 



24 THE PLANT CELL 

a drop and mount it under a coverglass, having first placed^ a 
hair in the drop to prevent the breaking up of the yeast colon- 
ies by the pressure of the coverglass. Study with a high 
power and draw a yeast colony showing different stages of 
budding. It would be interesting to note the development of 
the colonies from single individuals, and this can be done in 
a hanging drop culture. Pour some of the culture into a test- 
tube (2) and shake it vigorously to break up the colonies. 
Clean a coverglass thoroughly in soap and water, rinse it and 
rub it with a cloth dipped in alcohol. Place a drop of broth 
from test-tube ( 1 ) at the middle of the coverglass and dip the 
point of a needle first into the test-tube (2) and then into the 
drop to inoculate it with yeast. Now invert the coverglass 
over a hollow slide obtainable of the dealers, having run a 
thin film of vaseline around the border of the hollow for seal- 
ing the coverglass. Bring a yeast plant into focus with the 
high power and set the microscope in a warm place. Exam- 
ine the culture at intervals of half an hour or so. 

Instead of a hollow-ground slide a culture cell may be made 
by spinning a ring of melted paraffin on an ordinary slide. 



CHAPTER II 
DIFFERENTIATION OF THE TISSUES 

General Survey 

A tissue is a group of united cells that are essentially alike, 
have had a common origin and are prepared to perform a 
common function : thus, the outermost layer of cells, or epider- 
mis, of an apple; the groups of bast and wood fibers, the assem- 
blage of cells forming the pith, are tissues. At the growing 
apex of a stem there is but one tissue (Fig. n), for the cells 
are essentially all alike. This is known as the primordial 
meristem. 

On comparing cross and longitudinal sections at successive 
planes below the apex we find that the primordial meristem 
soon becomes changed into three distinct parts : the pro to derm 
at the exterior, the procambium strands, and the fundamental 
or ground meristem (Fig. 1 1 ) . These three regions or tissues 
are to undergo further differentiations, and are known as pri" 
mary meristems. In still older portions of the stem, lower 
down, we find that the protoderm has changed into a definite 
outer skin or epidermis; the procambium has become trans- 
formed into three parts, now collectively termed a vascular 
bundle, an outer part called the phloem, an inner, called the 
xylem, and a part between these two termed the cambium; and 
the ground meristem is seen to have become differentiated into 
four main parts, namely, primary cortex, pericycle; primary 
medullary rays, and medulla or pith (Figs, n and 14). These 
divisions are suggested by distinct landmarks ; thus, the inner 
limit of the primary cortex is a zone of cells, known as the 
endodermis or starch sheath, which is usually characterized by 
an abundant starch content ; the pericycle extends from this to 
the outer border of the vascular bundles; the primary medul- 

2 5 



26 



DIFFERENTIATION OF THE TISSUES 




1/ 



Cylem from the procambium 
Xylem from the cambium 
Phloem, from the procambium and cambium 



Xylem from the procambium 
Xylem from the cambium 
Phloem from the procambium and cambium 



Fig. ii. Diagram showing the evolution of tissues from the primordial meristem 
down to the beginning of cambial activity. In the longitudinal diagram, at the bottom, 
the initial C of the word cambium stands directly beneath this tissue which is radially 
but one cell in thickness. 



lary rays lie between the bundles, and are limited externally by 
the plane touching the outer borders of the phloem strands, 
and internally by the plane bounding the inner borders of the 
xylem strands; while the medulla or pith is all of the tissue 
surrounded by the bundles. Everything within the starch 
sheath is called the stele. 



THE PRIMORDIAL MERISTEM 2J 

The character of the different tissues and their origin and 
progress of development will now he considered. 

The Primordial Meristem. — The primordial meristem oc- 
curs at the growing apices, and from it all parts of the plant, 
directly or indirectly, take their origin. The cells composing 
it are nearly isodiametric, are essentially all alike, and are 
characterized by their relatively small size, relatively large 
nuclei, dense cytoplasm, absence of vacuoles, absence of insol- 
uble foods, such as starch and oil, very thin cellulose walls, and 
power of repeated division. They are of course very tender, 
having little strength and rigidity excepting that due to tur- 
gidity; and having in and of themselves no protection against 
loss of water they need to be protected when growing in the 
air, by the older tissues of scales, etc. The primordial meri- 
stem is continually carried forward by the enlargement of the 
cells beneath it that have been formed by its own cell division ; 
just as a man, standing on a stone wall which he is building, 
is carried upward with each successive course of stone. The 
cells and tissues are thus successively older as they recede from 
the apex. Some quality in the heredity of the daughter cells 
of the primordial meristem causes them soon to become differ- 
entiated into the three primary meristems called the protoderm, 
procambium, and ground meristem (Fig. n). 

The Protoderm. — This is the outermost layer of cells close 
to, and in some instances even surrounding the apex, which, 
after a period of cell division, is to become the epidermis. In 
most cases its cell divisions give rise to radial walls only. 
Radial walls run parallel with a line extending from center to 
circumference ; so that it increases in superficial expanse as the 
tissues within enlarge ; but in some instances it divides tangen- 
tially (that is, at right angles with the radius) and thus gives 
rise to a multiple epidermis of several cell layers. Usually the 
cells of the protoderm differ in form from those of the ground 
meristem bordering it within, but this is not always the case. 

The Procambium. — In dicotyledonous stems the procam- 
bium occurs as isolated strands disposed in the form of an 



28 DIFFERENTIATION OF THE TISSUES 

interrupted ring where later the vascular bundles appear. In 
cross sections the procambium cells are much smaller than 
those of the surrounding ground meristem; and in longitu- 
dinal sections they are seen to be elongated, and more or less 
pointed at the ends (Fig. n). In the evolution of a procam- 
bium strand from the primordial meristem groups of vertical 
rows of cells of the latter divide repeatedly by vertical walls, 
producing the small cells so plainly made out in cross sections ; 
these cells then elongate, as seen in longitudinal sections (Fig. 
n). They are then ready to differentiate into the different 
elements of the vascular bundles, and sometimes into bast 
fibers, as will later be shown. 

In the stems of Monocotyledons the procambium strands are 
scattered promiscuously instead of being disposed in a ring 
(Fig. 27), as in Dicotyledons; and in roots the procambium 
occurs as a single central strand. 

The Ground Meristem. — This has been derived from the 
primordial meristem by cross, as well as vertical, divisions of 
its cells. It is distinguished from the procambium by not 
being particularly elongated in any dimension, and from both 
procambium and protoderm by the relative largeness of its 
cells and the presence oftentimes of intercellular spaces. 

The cells of the primordial meristem retain the power of 
division throughout the life of the plant, but those of the 
primary meristems, namely, protoderm, procambium, and 
ground meristem, after a time cease to divide, excepting the 
cambium cells from the procambium, as will later be shown; 
and they then form what is known as the primary permanent 
tissues, the protoderm giving rise to the epidermis, the procam- 
bium forming the primary vascular bundles, and the ground 
meristem differentiating into the primary cortex, pericycle, 
primary medullary rays, and pith, as already stated. 

While these permanent tissues are characterized by the cessa- 
tion of cell division, yet in large groups of dicotyledonous 
plants the cells of the primary cortex habitually give rise by 
cell division to a zone of cork cambium or phellogen from 



THE GROUND MERISTKM 2(J 

which the cork is derived; and cells of the medullary rays in 
like manner produce cambium cells (interfascicular cambium I 
which, joining with the cambium of the vascular bundles 
(fascicular cambium) complete the cambium ring. The inter- 
fascicular cambium and the cork cambium are classed as sec- 
ondary mcristcm. The details of their origin and their rela- 
tion to the other tissues will appear under the discussion of 
secondary increase in thickness. 

The Primary Permanent Tissues. — The tissues formed by 
the transition of the primary meristems into the permanent 
state where cell division ceases are known as the primary per- 
manent tissues in contradistinction to those that are the prod- 
uct of cambium cells, which are called secondary permanent 
tissues. 

Beginning at the outside the primary permanent tissues in 
dicotyledons are grouped on anatomical and topographical 
grounds into the following main divisions: Epidermis, pri- 
mary cortex, pericycle, phloem part of the vascular bundle, 
xylem part of the vascular bundle, medullary ray, pith (Fig. 
14). In Monocotyledons the order is the same until the peri- 
cycle is passed, and then follow the vascular bundles, with 
phloem facing outward and xylem inward, scattered through 
the ground tissue, where, in consequence, medullary rays and 
pith are not distinguishable (Fig. 28). The tissue groups in 
dicotyledons will now be taken up in the order named. 

Epidermis. — As the protoderm cells are becoming trans- 
formed into the epidermis they enlarge more or less, varying 
a great deal in this respect in different plants and habitats, and 
in different parts of the same plant ; and they assume a diver- 
sity of forms, from isodiametric to much elongated, and from 
regular polyhedra to forms of sinuous contour (Fig. 12) ; and 
they may even grow out from the surface in the form of scales 
and hairs. 

While the changes in size and form are going on, the outer 
wall is as a rule thickening and becoming chemically altered, 
particularly in parts exposed to the air. The most important 



30 



DIFFERENTIATION OF THE TISSUES 



chemical change is due to the addition and infiltration of a 
waxy substance called cutin which makes the outer wall water- 
proof. This occurs in its greatest purity as a thin film at 
the outer surface known as the cuticle. In some instances 

scales and rods of wax are 
deposited at the surface and 
cause the so-called bloom, as 
on the plum, grape, stems 
of some grasses, etc., where 
they are easily rubbed off on 
account of their delicacy. . In 
sections under the micro- 
scope the wax, cuticle, and 
cutinized parts of the wall 
are readily distinguishable 
by their yellow color when 
treated with chloroiodide of 
zinc, while the cellulose por- 
tions of the wall assume at 
the same time a purplish 
color. 

The thickening and cutini- 
zation of the outer wall 
makes of the epidermis an 
excellent protective tissue 
against loss of water, para- 
sitic fungi, and, to a certain 
extent, mechanical injury. 
How quickly, for example, 
an apple will begin to dry up and decay when its epidermis is 
pared away. 

The cells of the epidermis remain living so long as they are 
not cut off from the water supply by the formation of cork 
tissue. Their protoplasts exist as a very thin and hardly dis- 
tinguishable film lining the walls. In rare instances, particu- 
larly in the Monocotyledons, the epidermal cells contain chloro- 




Fig.. 12. i, epidermis of oak leaf; 2, 
epidermis of Iris leaf, both viewed from 
the surface; 3, group of cells from petal 
of Viola tricolor; 4, two epidermal cells in 
cross-section showing thickened outer wall 
differentiated into three layers, namely, an 
outer cuticle, cutinized layer (shaded), and 
an inner cellulose layer; 5 and 6, epidermal 
outgrowths in the form of scales and hairs. 
(3 after Strasburger, 4 after Sachs, and 5 
after de Baryj 



THE PRIMARY PERMANENT TISSUES 



31 




plasts; and sometimes, as in fruits and foliage plants, a colored 
cell sap; but in most cases they are colorless and permit the 
unimpeded entrance of light. While they live the epidermal 
cells are filled with cell sap, largely water, and serve in many 
cases as an effective storehouse for water. This function is 
carried to its fullest development where 
a multiple epidermis is produced by the 
tangential division of the protoderm, as 
in the rubber leaf (Fig. 13). 

In herbaceous plants, and in leaves gen- 
erally and fleshy fruits the epidermis re- 
mains, but in the perennial parts of plants 
that increase in size from year to year, 
such as the steins and roots of trees and 
shrubs, and in many underground parts 
that endure but for a season, such as the 
Irish potato, cork is after a time produced 
beneath the epidermis and this dies and 
sloughs away. 

Primary Cortex. — All of the ground 
meristem lying exterior to the procam- 
bium strands (which, it will be remembered 
later become the primary vascular bundles) produces perma- 
nent tissues that are divisible into two zones by a layer of cells, 
more or less continuous, known as the starch sheath or endo- 
dermis. The outer of these zones, which includes the starch 
sheath, is known as the primary cortex (Fig. 14). (The 
term primary cortex must not be confused with cortex as used 
in pharmacognosy, where cortex, employed synonymously 
with bark, is often applied to all of the tissues collectively 
which lie exterior to the cambium ring. ) The primary cortex 
does not as a rule consist of a single tissue, but of two or 
more, so that in describing its evolution from the ground meri- 
stem the possible tissues composing it must be considered 
separately. 

Beginning at its exterior just beneath the epidermis we com- 



Fig. 13. Cross-section 
of a portion of leaf of 
Ficus elastica showing the 
multiple epidermis from 
e to a inclusive; c, cysto- 
lith; b, palisade paren- 
chyma; d, spongy paren- 
chyma. (After Sachs.) 



32 



DIFFERENTIATION OF THE TISSUES 




Cambium 



Fig. 14. Diagram to show the topography and character of the tissues that are evolved 
from the primary meristems. Cambial activity has not yet begun. 






TIM. PRIMARY PERMANENT TISSUES 33 

monly find a tissue whose walls are thickened at the angles 
where three or four cells join. This tissue is called the col- 
lenchyma (Fig. 14). It is one of the first of the primary 
tissues to come to maturity, and its chief function, in virtue 
of its thickened walls, is to give strength at a time when the 
bast and wood fibers have not yet made their appearance or 
arrived at sufficient maturity to be effective. It may occur as 
a continuous zone or in separate strands. In producing the 
collenchyma the ground meristem cells divide transversely and 
vertically, the daughter cells enlarge and elongate vertically; 
the walls gradually thicken at the angles, but the cellulose com- 
posing them does not appear to become essentially altered or 
replaced. The collenchyma cells usually contain chloroplasts, 
and they remain living until cut off from the general circula- 
tion by cork cells in the formation of borke (see page 57), as 
frequently happens in woody plants. 

Thin-walled Parenchyma lies next to the collenchyma inter- 
nally, and as a rule constitutes most of the primary cortex. 
It has been evolved from the ground meristem by transverse 
and vertical division of the cells of the latter, and the growth 
of these cells about equally in all dimensions, or with a slight 
excess of elongation in the vertical or radial direction (Fig. 
14). The cell-walls of this tissue remain thin, and their orig- 
inal cellulose is practically unchanged. The cells frequently 
contain chloroplasts and remain alive unless involved in the 
formation of borke, as stated for collenchyma. In virtue of 
its chloroplasts this tissue, as well as the collenchyma, is able 
to manufacture food, and it is much used also in the slow con- 
duction and storage of foods that have come to it from the 
leaves. 

Short Sclerenchyma Cells or Stone Cells are frequently 
found, singly or in groups, distributed amongst the thin-walled 
parenchyma (Fig. 14). These, as a rule, are transformed 
thin-w r alled parenchyma cells whose walls have become greatly 
thickened and more or less lignified. The tubular and often 
branched pits characteristic of the walls of these cells are thin 

4 



34 



DIFFERENTIATION OF THE TISSUES 



places left as the thickening of the walls progresses. These 
cells soon die after the completion of their walls and seem 
chiefly to be used in giving strength, and protection. 

Long Sclerenchyma Celts or Bast Fibers sometimes occur 
in the primary cortex (Fig. 14). They take their origin from 
procambium strands that have arisen from vertical division 
of the cells of the ground meristem or protoderm. In the 
latter instance they would be classified with the epidermis from 






< 





Fig. 15. Diagram showing stages in the development of collenchyma, stone cells, 
and bast fibers. A, collenchyma; i and 2, cross and longitudinal section of a collen- 
chyma cell in its primary meristem condition; 3 and 4, longitudinal and cross-sections 
of the same cell at a later stage, the walls in 4 have commenced to thicken at the 
angles; 5 and 6, longitudinal and cross-sections of a mature cell. The arrows indicate 
the planes through which the longitudinal sections were cut. The stippling inside 
the walls indicates the protoplasts. B, stone cells; 1, in the primary meristem condi- 
tion; 2, the cells have enlarged and the walls have begun to thicken and become 
pitted; 3, the walls are completed. The primary wall is black, cellulose additions 
white, and the lignified walls in 3 are stippled. Notice that the protoplasts have 
disappeared in 3, and the pits in some instances are branched; C, bast fibers; 1 and 2, 
cross and longitudinal sections of primary meristem cells that are to become bast 
fibers; 3 and 4, the same at a later stage; 5, longitudinal section of completed bast 
fibers. In 5 the stippling of the wall indicates lignification. Note that the walls 
have become pitted and the protoplasts have disappeared from the fibers. 



THE PRIMARY PERMANENT TISSUES 



35 




Fig. i 6. 
section of 
ascalonicum. 



the standpoint of their origin ; but on account of their position 
they will here be included with the primary cortex. In their 
procambium state the bast fibers are thin- and cellulose-walled 
and vertically elongated ; proceeding toward maturity the pro- 
cambium cells enlarge, and principally in the vertical direction, 
so that the ends shove past each other and become pointed ; 
the walls thicken and become lig- 
nified as a rule (Fig. 15), and the 
protoplasts finally disappear, leav- 
ing the fibers dead. This is the 
mode of origin of bast fibers wher- 
ever they occur. 

The bast fibers seem to serve 
chiefly, if not solely, for giving 
strength, for which purpose they 
are fitted, whether they occur in 
the primary cortex or in the peri- 
cycle, by their vertical elongation, 
thick and lignified walls, and dove- 
tailing or interlacement of the 
ends. In roots the primary cortex 
usually consists of thin-walled parenchyma alone (Fig. 16). 

The innermost layer of the primary cortex is the starch 
sheath or endodermis (Fig. 14). This in stems is, as its 
name implies, unusually rich in its starch content, and other 
than this, in stems, it oftentimes has no striking characteristics. 
In recent years good evidence has been brought forward, 
notably by Haberlandt, to show that the starch grains in this 
tissue act like the otoliths in the ear, and by falling always 
to the lower side of the cell as the position of the part to 
which they belong is shifted, they furnish by their impact the 
stimulus for perceiving the direction of the gravity pull. 

In other instances, and particularly in roots, the endodermal 
cells become differentiated from the rest of the primary cortex 
by elongating somewhat in the vertical direction, suberizing 
their radial walls, and by partially or completely thickening 
their Avails (Fig. 16). 



m 
n 

Portion of a cross- 
t root of Allium 
h, large central 
tracheal tube; i, xylem and /, 
phloem portion of the vascular 
bundle; n, cortex cells; j, endo- 
dermis with thin-walled cell at 
k to admit passage of materials; 
m, pericycle or pericambium. 
(After Haberlandt.) 



3^ DIFFERENTIATION OF THE TISSUES 

The intercellular spaces that can be found in other tissues 
of the primary cortex are lacking in the endodermis. These 
characteristics have led to the conception that the endodermis 
possessing them is intended to reduce permeability between 
primary cortex and stele; and this conception is strengthened 
by the occurrence, in such an endodermis, of thin-walled cells 
just in front of the xylem portion of roots where water ab- 
sorbed from the soil has need of access to the water tubes (Fig. 
16). In old portions of roots it often happens that the outer 
parts of the primary cortex slough away, leaving the endoder- 
mis to protect the stele. 

The Pericycle. — The pericycle lies between the starch sheath 
or endodermis and the outer rim of the phloem part of the 
vascular bundles (Fig. 14). In stems we commonly find it 
composed of two kinds of tissues, thin-walled parenchyma, 
and bast fibers, the origin of which from the ground meristem 
is as stated for the corresponding tissues in the primary cortex. 




ABC 

Fig. 17. Diagram to show different plans in the distribution of bast fibers. A, 
bast a continuous cylinder in the pericycle; B, isolated strands of bast in the cortex 
and in the pericycle in front of each vascular bundle; C, a combination of A and B. 
(After Green.) 

The bast fibers may form a continuous zone all around the 
stem, or they may occur as isolated groups, either associated 
with, and seemingly a part of the phloem of the bundles, or 
dissociated from the phloem (Fig. 17). In the stems of most 
dicotyledonous plants the bast fibers are restricted to the peri- 
cycle. They serve, of course, for giving strength ; and unless, 
or until, the cambium later adds a substantial amount of wood 
fibers or fiber tracheids they remain the chief reliance in this 
respect. 



nil; PRIMARY PERMANENT TISSUES 37 

The thin-willed parenchyma cells of the pericycle, like those 
of the primary cortex, often contain chloroplasts, and they 
serve for the slow conduction and storage of reserve foods, 
particularly of the non-nitrogenous class. 

In roots the pericycle occurs usually as a single layer of 
thin-walled cells (Fig. 16), and its chief significance here is 
that from the division of its cells the lateral roots take their 
origin. 

The Primary Vascular Bundle. — The typical vascular bundle 
of dicotyledonous stems consists, as already stated, of three 
parts, an outer or phloem, an inner or xylem, and a median or 
cambium. These, in primary bundles, are all the product of 
the differentiation of the procambium, and the progress of 
their evolution will now be followed. 

A typical phloem consists of three elements, the sieve tubes, 
the companion cells, and the sieve or phloem parenchyma (Fig. 
14). A sieve tube consists of a vertical row of cells each of 
which is vertically elongated and separated from its neighbor 
above and below by a thickened partition wall that is perfor- 
ated by many openings. These partition walls somewhat re- 
semble a sieve and have therefore suggested the name for the 
tube (Fig. 14). 

In the evolution of a sieve tube a vertical row of procam- 
bium cells divides longitudinally, producing a double vertical 
row of cells. The cells of one row enlarge transversely and 
vertically; their transverse or end walls thicken, leaving thin 
places or pits, and these finally become complete, opening from 
one cell cavity to another by absorption of the wall at the bot- 
tom of the pits, and the vertical row of cells in this way 
becomes a continuous tube (Fig. 18). 

The vertical walls of the sieve tubes are usually compara- 
tively thin, but they are sometimes markedly thickened. The 
cells in the row companion to the sieve tubes, known as the 
companion cells, enlarge in all dimensions somewhat, but re- 
main much smaller than the cells of the sieve tubes. The cells 
of the sieve tubes remain alive, at least throughout the first 



38 



DIFFERENTIATION OF THE TISSUES 



abcdb 

mm 

IP iff 




year of their formation, but they strangely lose their nuclei. 
This is an anomaly, for cells that have been deprived of their 
nuclei artificially soon die. It is thought in this instance that 
the nuclei of the companion cells extend their influence to those 

of the sieve tubes and so keep up 
there the oxidative and other 
processes that depend upon nu- 
clear activity. The walls of both 
sieve tubes and companion cells 
remain cellulose. 

The contents of the sieve tubes 
are found to be rich in proteids, 
amido-acids, and soluble carbohy- 
drates, and minute starch grains 
may sometimes be present in abun- 
dance. Even proteids that are 
in solution do not pass readily 
through cell walls, and in the sieve 
tubes the perforations allow them 
to pass in an unobstructed stream 
from one cell or sieve tube mem- 
ber to the other. That the sieve 
tubes are for the vertical flow of 
proteids and allied substances is 
shown by direct observation under 
the microscope while using suita- 
ble reagents for the demonstration 
of proteids ; and further by gird- 
ling and constriction experiments 
- described in Chapter X. 
The sieve parenchyma cells in differentiating from the pro- 
cambium elongate vertically more or less and increase in their 
cross diameters (Fig. 18), but they do not become as a rule 
so large in any dimension as the cells of the sieve tubes. Their 
walls remain cellulose and commonly thicken but little. They 
appear to serve chiefly in the translocation of carbohydrates; 



Fig. i 8. Stages in the devel- 
opment of sieve tubes, companion 
cells and phloem parenchyma. A, 
a and b, two rows of procambial 
cells; in c and d, a has divided 
longitudinally and c is to become 
companion cells; d, a sieve tube, and 
b, phloem parenchyma ; B, c, compan- 
ion cells, and d, a beginning sieve 
tube from c and d respectively in A 
The cross-walls in d are pitted; b, 
phloem parenchyma grown larger 
than in A; C, the same as B with 
the pits in the cross-walls of the 
sieve tubes become perforations, and 
the nuclei gone from the cells com- 
posing the tube. 



THE PRIMARY PERMANENT TISSUES 39 

and as sti »rage places for proteids which they are in position to 
take from the sieve tubes when a surplus is at hand, and they 
assist in delivering over to the medullary rays materials from 
the sieve tubes to be stored by the rays or transported inward 
for storage in the cells of the wood or xylem parenchyma. 

The sieve tubes, companion cells, and sieve parenchyma cells 
seem to remain alive and functional throughout the first year 
of their origin, and in some instances certainly for several 
years, but it may be stated as a rule that they soon become 
crushed by the pressure of surrounding tissues, and their effec- 
tiveness is thereby reduced, and after a time altogether de- 
stroyed (Fig. 24). 

The three kinds of cells taking part in the formation of the 
phloem as described above do not always occur together; in 
Monocotyledons, for example, the parenchymatous elements 
are absent, and in Gymnosperms and Pteridophytes the com- 
panion cells are lacking. 

The primary xylem (namely, that portion of the xylem that 
has differentiated from the procambium and exclusive of that 
which is added later by the cambium) may consist of three 
classes of elements, the tracheal or water tubes, tracheids, 
xylem parenchyma, and wood fibers. These elements do not, 
however, commonly all occur together. The wood fibers are 
usually absent, and tracheids are not common in the primary 
xylem of Angiosperms; while in Gymnosperms true tracheal 
tubes do not occur, with few exceptions. 

A tracheal tube is produced by the absorption of the end or 
transverse walls in a vertical row of cells, and at the same time 
the enlargement in all dimensions of the cells composing the 
tube, and the subsequent unequal thickening and lignification 
of the vertical walls. The thick places in the walls are for 
strengthening the tube, while the thin places are for the easy 
passage in and out of water and materials in solution. In the 
tubes first formed the thick places are in the form of rings or 
a spiral coil. To realize the use of these, imagine barrel hoops 
or a flexible spiral coil of wood sewed inside a bag to make it 



40 DIFFERENTIATION OF THE TISSUES 

stand open. This type of tracheal tube is differentiated from 
the procambium not far from the growing apex in internodes 
that have not yet ceased elongating, and it will be seen that 
the corresponding growth in length which these tubes must 
undergo will be but little resisted by the kind of thickenings 
which their walls possess. In older internodes where elonga- 
tion has nearly or quite ceased stronger tracheal tubes are laid 
down having thin places of more restricted area in the form of 
pits or elongated meshes. 

While a tracheal tube consists of a chain of fused cells a 
tracheid is a single cell only with thin places in its walls in the 
form of pits with overhanging border for greater strength. 
These are known as bordered pits (Fig. 19). Or the thicken- 
ings may be of the spiral and reticulate types. In becoming 
a tracheid the procambium cell elongates and tapers at the end 
to a greater or less degree, thickens its wall unequally, and 
finally lignifies its wall (Fig. 19). 

The tracheal tubes are primarily for carrying water from 
roots to and throughout the leaves ; and the tracheids have the 
same function, but they may assume the character of wood 
fibers and be depended on for strength as well as for conduct- 
ing water, as in the case of pine wood. 

The cells of the xylem parenchyma are, as a rule, relatively 
thin-walled, and the walls are sometimes, and in woody plants, 
commonly, lignified, and they may or may not be pitted. To 
form the xylem parenchyma the procambium cells divide 
transversely, and the young parenchyma cells enlarge in all 
dimensions, becoming more or less elongated vertically, with 
end walls at right angles to the vertical or, as a rule, only 
slightly inclined (Fig. 19). They differ from the tracheids 
by their less elongation, blunter ends, commonly thinner walls 
and unbordered pits. The proportion of xylem parenchyma to 
the other elements of the primary xylem varies greatly in dif- 
ferent examples, from occupying the bulk of the xylem to 
entire absence. Its function is to store reserve water and 
foods, and possibly to assist in lifting the water to the leaves. 



THE PRIMARY PERMANENT TISSUES 4 1 

The wood fibers are characterized by being much elongated 
and taper-pointed at the ends, and by thick and lignified walls 
and small unbordered pits. The steps in the evolution of a 
wood fiber from a procambium cell are evident. The cell elon- 



H 



:d 



i) 



© 
© 




I© 



@ 

© 




® 




P 




m 





"'■ *W?" " 




r ] 




!?f>.:!r>.'<;-0-:j.Y.:itf.>j 




,"*«aa*"--n 


4 


,'.. ~;*tew ■ 


s 


^, 1 



D 

Fig. 19. Stages in the development of the elements of the xylem. A, progressive 
steps in the development of a tracheal tube, i, row of procambial or cambial cells 
that are to take part in the formation of a tube; 2, the same at a later stage enlarged 
in all dimensions; 3, the cells in 2 have grown larger, their cross-walls have been 
dissolved out, and the wall has become thickened and pitted; 4, the walls in 3 have 



4 2 DIFFERENTIATION OF THE TISSUES 

gates, and in doing this the ends of contiguous cells shove past 
each other; the walls gradually thicken, and finally become 
more or less completely lignified. The wood fibers are chiefly 
to give strength, and they are assisted in this by their inter- 
lacing and dove-tailing together, as well by their thick and 
lignified walls. 

The cells composing the tracheal tubes soon die, so that the 
tube is not long alive after it comes to maturity. The wood 
fibers may live but one or only a few years, while the tracheids 
in respect of length of life seem to vary between the tracheal 
tubes and the wood fibers. The parenchyma cells are, as a 
rule, the longest lived of all xylem elements. They have been 
found still carrying on the vital function of starch storage in 
rings of growth nearly a century old. 

After the elements of the primary xylem and phloem have 
been completed it is found that a layer of undifferentiated pro- 
cambium cells remains between them in Dicotyledons, while in 
Monocotyledons and Pteridophytes the whole of the procam- 
bium enters into the composition of phloem and xylem. The 
layer of undifferentiated cells in Dicotyledons is known as the 
cambium, and its most important characteristic is that it retains 
the power of cell division for a longer or shorter time, even 
indefinitely in the case of woody perennials. 

The divisions of the cambium cells may take place trans- 
versely, or vertically in radial and tangential planes; but the 

become more thickened, the pits have an overhanging border, the walls have become 
lignified as indicated by the stippling, and finally the protoplasts have disappeared, 
and the tube is mature and dead; B, stages in the formation of tracheids from pro- 
cambial or cambial cells. The steps are the same as in A, excepting that the cross- 
walls remain and become pitted. C, steps in the development of wood fibers from 
cambial cells; i, cambial cells; 2, the same grown larger in all dimensions with cells 
shoving past each other as they elongate; 3, a later stage with cells longer and more 
pointed and walls becoming thickened and pitted; 4, complete wood fibers with walls 
more thickened than in the previous stage and lignified, as shown by the stippling. 
The protoplasts in this last stage have disappeared and the fibers are dead. D, steps 
in the formation of wood parenchyma from cambial or procambial cells. 1, group of 
cambial or procambial cells; 2, the same enlarged in all dimensions; 3, the same with 
walls thickened and pitted; 4 and 5 show the same stages as 2 and 3, but here the 
cells have enlarged radially or tangentially more than they have vertically. The 
walls of these cells are apt to become lignified, but the cells are longer lived than 
the wood fibers. 



THE PRIMARY PERMANENT TISSUES 43 

tangential vertical division is by far the most frequent and 
gives rise to cells known as the tissue mother-cells, which by 
one or more cell divisions produce other cells that differentiate 
into the elements of the xylem on the one hand and those of 
the phloem on the other. These new additions to the primary 
vascular bundle constitute the secondary xylem and phloem. 

The medulla or pith is formed by transverse and vertical 
divisions of the ground meristem, and the subsequent enlarge- 
ment in all dimensions of the daughter cells. The pith cells 
are not apt to be much elongated in any one dimension, and 
their walls, as a rule, remain relatively thin and unchanged 
from their original cellulose composition; but they are some- 
times decidedly thickened and lignified. They are short lived ; 
in woody plants the surrounding bundles crowd in and crush 
them, and in herbaceous plants they often break down and 
leave a hollow space; in other cases still they may persist for 
a long time as dead elements. The pith cells are for some time 
of use in the storage and slow conduction of water and some- 
times they are employed in the storage of food, even after 
they are several years old, but they are evidently not commonly 
depended on long for any essential function. 

The ground meristem lying between the vascular bundles 
undergoes cell division vertically and transversely and the cells 
thus formed enlarge and frequently become elongate in the 
radial direction. In this way the primary medullary rays are 
formed. The medullary rays have two distinct functions: 
they carry water and substances in solution radially, inward 
and outward as needed, and they store water and reserve foods. 
Since the primary rays extend usually only a few millimeters 
vertically they are not suited for transporting materials in that 
direction. A study of cross-sections needs to be supplemented 
by an examination of vertical tangential sections to make the 
extent of the medullary rays clear. In these vertical sections 
it is seen that the primary vascular bundles do not maintain an 
independent and isolated course through the stem, but anasto- 



44 



DIFFERENTIATION OF THE TISSUES 




mose with each other across the upper and lower borders of 
the rays, as seen in Fig-. 20. 

In the foregoing a type of vascular bundle has been chosen 
known as the collateral type; where one phloem strand stands 
radially in front of the xylem strand. While 
this is the prevailing type there are others 
that must not be passed unnoticed here. It 
sometimes happens that a second phloem 
strand stands radially within, or on the pith 
side of the xylem, forming what is known 
as the bicollateral bundle; in other instances 
the phloem surrounds the xylem, or vice 
versa, making a concentric bundle; while in 
roots it is the rule that the phloem and xylem 
of the primary bundle are in strands alternat- 
ing with each other, neither standing radially 
in front of the other, thus making what is 
called a radial bundle. (See Fig. 21 for 
these types.) 

In leaves the primordial meristem becomes 
differentiated into protoderm, ground mer- 
istem and procambium strands; and, just as 
in stems, the protoderm gives rise to the epidermis, the 
procambium strands to the vascular bundles which make up 
the greater part of the veins, and the ground meristem to 
the mesophyll cells, which correspond to the primary cortex of 
stems. Near the bases of leaves, below where the vascular 
bundles have become split up to form the smaller veins, there 
is often a sheath of cells surrounding the bundles which corre- 
sponds to the parenchyma cells of the pericycle of stems. 

Where the vascular bundles first enter the leaves they have 
essentially the same constitution as in stems, with the excep- 
tion of a functional cambium, but they become smaller as they 
proceed and ramify throughout the leaf and have correspond- 
ingly fewer parts. The rule is that the sieve tubes gradually 
give place to elongated but otherwise undifferentiated paren- 



Fig. 20. Diagram 
showing how the vas- 
cular bundles anas- 
tomose around the 
medullary rays. The 
gaps represent the 
rays. 



ILLUSTRATIVE STUDIES 



45 



chyma cells; and in the ultimate ramifications only spirally 
and reticularly thickened tracheids are left to represent the 
bundle, and these are surrounded by a sheath of parenchyma 
cells belonging to the mesophyll or primary cortex, and known 




Fig. 21. Different types of vascular bundles. A, the concentric type, with xylem, 
k, surrounding the phloem, h. B, the collateral type, with phloem, h, standing in 
front of the xylem, k. C, a portion of the radial type, shown complete in D, where 
the part outlined at a, corresponds to C. Corresponding parts are lettered the same in 
both figures; c, xylem; b, phloem; f, cambium ring; e, pericycle; d, endodermis. C 
and D are from the tap root of Vicia faba. (After Haberlandt.) 

as the parenchyma sheath. It is the function of these meso- 
phyll cells to collect and carry toward the base of the leaf the 
foods manufactured by the rest of the mesophyll (see Fig. 90). 



Illustrative Studies 

1. Prepare cross and longitudinal sections of the growing 

apex of stems of Aristolochia sipho by imbedding the material 

in paraffin and making permanent stained mounts as described 

under Cytological Methods in Chapter XIII. Use erythrosin 



46 DIFFERENTIATION OF THE TISSUES 

and iodine green for the stains or safranin and hematoxylin. 

Prepare in the same way sections from several successive 
internodes back from the apex. The object is to follow the 
progressive development of the tissues from the primordial 
condition at the apex down to where the primary permanent 
tissues appear. Find all of the tissues described in the 
chapter. 

Draw a few cells from each tissue, using the eyepiece scale 
(page 246) to get all details to scale. Determine the actual 
sizes of the cells and thicknesses of the walls. 

The erythrosin and iodine green will stain cellulose walls 
pink, and lignined and cutinized walls green, and in this way 
it can be determined how far back from the apex the original 
cellulose walls first become modified for specific purposes. 

Notice where the first elements of the vascular bundle ap- 
pear in the procambium and how in older segments of stem 
these have become longitudinally stretched. 

2. Make cross and longitudinal sections of Aristolochia 
stem where the stem is older than where the sections in the 
above studies were taken, but where the cambium has not yet 
begun to add to the thickness of the stem. The stem in this 
region will probably be too hard for the paraffin process and 
the sections may be cut free-hand or on a sliding microtome 
as described on page 220. Double stain the sections in ery- 
throsin and iodine green and make permanent mounts in bal- 
sam (page 232). Note the changes which each tissue has 
undergone since the earlier stages and make drawings to scale 
to show these changes. Think over carefully what you have 
seen and embody your results in your permanent note-book. 

If it is thought best not to make the permanent mounts as 
above suggested the sections can be examined in chloroiodide 
of zinc (page 259) in which cellulose walls will be blue and all 
others yellow. One objection to this reagent is that it swells 
the walls more or less. In its place aniline sulphate might be 
used (page 254) and then the lignified walls would be yellow 
and all others would be uncolored. Cutinized walls could 



ILLUSTRATIVE STUDIES 47 

then be demonstrated by leaving sections for several hours in 
alcanna tincture (page 252) when these walls would be pink. 

3. Note the functions that in this chapter are attributed to 
each tissue, and see in what ways the tissues are adapted to 
them. Enter your observations in your permanent note-book. 

4. Make a cross-section of a leaf through one of the lateral 
veins and identify there the epidermis and the vascular bundle 
of the vein. The rest of the tissues belong to the fundamental 
or ground tissue, called in the leaf the mesophyll. Since the 
leaf is to be studied in detail in another place it will suffice 
here to enter in the note-book a simple diagrammatic drawing 
showing the relative positions and amounts of these different 
tissues. 



CHAPTER III 

SECONDARY INCREASE IN THICKNESS 

Dicotyledons and Gymnosperms 
If we follow the history of any particular region of stem 
or root we find that its growth in thickness up to the time 
when its growth in length ceases is due to the enlargement of 
the cells that arise from the division of the cells of the pri- 
mordial and primary meristems. In other words, increase in 
thickness is at first due to the enlargement of the cells of the 
epidermis, primary cortex, pericycle, phloem, xylem, medullary- 
ray, pith. Such increase, known as primary increase in thick- 
ness, soon ceases, and subsequent growth in thickness is due to 
the differentiation of additional tissues following the produc- 
tion of new cells by the division of the cambium, or cork 
cambium in the bark. 

Growth of the Vascular Bundles. — It will be remembered 
from the preceding chapter that the cambium ring is composed 
of two parts : the fascicular cambium consisting of procam- 
bium cells lying between the phloem and xylem which remain 
practically unchanged in form and retain their power of divi- 
sion, and the interfascicular cambium which is formed by the 
tangential division of primary medullary ray cells that lie in 
a line connecting the fascicular cambium of contiguous bun- 
dles. The cambium cells begin active cell division immediately 
following the differentiation of the primary tissues told about 
in the last chapter, and by the differentiation of these new cells 
the fascicular cambium adds tissues to the xylem toward the 
inside, and to the phloem toward the outside, and the inter- 
fascicular cambium makes additions in like manner to the pri- 
mary medullary rays. 

It is found on comparing the rate of growth of phloem and 

48 



GROWTH OF VASCULAR BUNDLES 



49 



fclZ* * 




Fig. 22. Diagram showing additions to the primary tissues through the activity 
of the cambium and phellogen or cork cambium. Compare this with Fig. 14. In this 
diagram stone cells have been omitted. 



5o 



SECONDARY INCREASE IN THICKNESS 



xylem that the latter increases much more rapidly than the 
former. This is due to the fact that when a cambium cell 
divides by the formation of a tangential wall, which it usually 
does, the daughter cell facing the xylem much more frequently 
differentiates into the permanent condition than the one facing 
the phloem, the latter continuing as a cambium cell ; but some- 
times the daughter cell facing the phloem grows to be one of 
the phloem elements while the one facing the xylem remains 
in the cambium condition. 

The kinds of tissues which the cambium adds to the xylem 
vary in different plants. In many Gymnosperms wood paren- 
chyma cells are formed, but, except in a single genus, 

neither tracheal tubes 
nor wood . fibers, their 
place being usurped by 
tracheids which per- 
form alike the strength- 
ening and water-con- 
ducting functions. In 
Angiosperms are pro- 
duced tracheal tubes of 
the pitted type, tra- 
cheids, and transitional 
forms between these 
two, xylem parenchyma 
and wood fibers, and 
transitional forms be- 
tween these also (Fig. 
22). On the phloem 
side the cambium adds 




Fig. 23. Photomicrograph of cross-section of 
stem of Aristolochia sipho, where cambial activity 
is just beginning, a, epidermis; b, collenchyma; c, 
thin-walled parenchyma of the cortex, the innermost 
cell layer of which is the starch sheath or endo- Sieve tubes, and, Vary- 
dermis; d sclerenchyma ring of the pericycle; . j ^ ^ kind f 

thin-walled parenchyma of the pericycle; f, pri 
mary medullary ray; g, phloem; h, xylem; i, inter 
fascicular cambium; j, medulla or pith, x 40. 



ing 

plant, companion cells 
or phloem parenchyma, 
or both of these, and, in many instances, bast fibers. 

While, by its tangential divisions, the cambium is thus adding 



GROWTH OF VASCULAR BUNDLES 51 

to the radial diameters of the phloem and xylem it is also, but 
at a slower rate, increasing their tangential diameters by its 
radial divisions ; so that, in cross-sections, the vascular bundle 
lias the form of a wedge with its apex pointing towards the 
center (Figs. 22, 23 and 24). As this wedge broadens new 
or secondary medullary rays are from time to time begun by 
the fascicular cambium (Fig. 24). These rays average less 
than half a millimeter in vertical extent, although in a few 
instances they run 100 to 200 millimeters from node to node; 
and in their tangential diameter they are seldom more than 
five hundredths of a millimeter, while radially they keep pace 
in growth with the phloem and xylem, and so always extend 
from the place of their origin in the xylem out between the 
phloem strands. The stimulus to form more medullary rays 
seems to come from the need of more radial highways as the 
diameter of stem and root and absorbing surfaces of new 
roots and food-building tissues of new leaves increase. The 
details of this will be discussed in Chapters VII and X. The 
daughter cells of the fascicular cambium, in becoming secon- 
dary medullary ray cells, enlarge chiefly in their radial and 
tangential diameters, becoming elongate radially as a rule in 
the xylem, but often vertically in the phloem. 
• While the vascular bundle is thus enlarging and secondary 
medullary rays are being laid down, the interfascicular cam- 
bium is adding new cells to the primary medullary rays and 
thus causing them to keep pace in radial growth with the vas- 
cular bundles. Not infrequently, however, the interfascicular 
cambium forms new vascular bundles which, in cross-section, 
appear to cut the primary ray into narrow strips. 

In the xylem portion of both primary and secondary medul- 
lary rays the tangential walls remain relatively thin or become 
pitted, and the radial walls are thin or pitted where they come 
into contact with tracheal tubes, tracheids, or xylem paren- 
chyma ; while the transverse walls are apt to be much thickened 
and lignified. In the phloem portion of the rays the walls 
remain thin and unlignified. 



52 



SECONDARY INCREASE IN THICKNESS 



Fig. 24 shows that the cambium adds much more to the 
xylem than it does to the phloem. The growth of the xylem 
continues as a rule to the middle or end of August, but the 
growth of the phloem continues after this even until frost. 

The kinds and relative amounts of the tissues in secondary 
xylem and phloem vary a great deal in different families, gen- 
era, and species ; and this fact is often very useful to the anato- 
mist and pharmacognosist in characterizing and identifying 
materials. Thus, the 
xylem parenchyma 
may vary from abun- 
dance to entire ab- 
sence; tracheidsmay 
prevail or be lack- 
ing ; tracheal tubes 
may vary greatly in 
size and number, or 
in their contact with l 
one another or com- 
plete isolation ; wood 
fibers may be pres- 
ent or absent, numer- 
ous or infrequent. 
The character of the 
phloem may vary in 
like degree; bast 
fibers may or may 
not occur; and so 
with the companion 
cells and phloem 
parenchyma. 

One of the most 
wonderful things 
about plants is that 
the daughter cells of 
the cambium may 




Fig. 24. Portion of cross-section of four-year-old 
stem of Aristolochia sipho, as shown by the rings of 
growth in the wood. The letters are the same as in 
Fig. 23, but new tissues have been added by the activity 
of the cambium; and a cork cambium has arisen from 
the outermost collenchyma cells and given rise to cork. 
The new tissues are: /, cork cambium; k, cork; g, sec- 
ondary phloem from the cambium, and just outside this 
is older crushed phloem; n, secondary xylem produced 
by the cambium; m, secondary medullary ray made by 
the cambium (notice that this does not extend to the 
pith). Half of the pith, h, is shown. Notice how it has 
been crushed almost out of existence. Compare Figs. 
23 and 24, tissue for tissue, to find out what changes 
the primary tissues undergo with age, and to what 
extent new tissues are added. Photomicrograph x 40. 



GROWTH OF VASCULAR BUNDLES 5 3 

become such various things. How is it that a daughter cell 
facing outward is directed to take part in the formation of a 
sieve tube, while one facing inward undergoes quite other trans- 
formations to form a tracheal tube ? And how can the daughter 
cells facing inward become any one of the xylem elements, 
apparently quite at will? They all have the same parentage, 
and probably the same potentialities, but behave differently, 
possibly because they are responding to stimuli of different 
natures. It may be that these stimuli arise in present need or 
necessity; but the response is modified by the peculiar poten- 
tialities of the particular species. Thus, the daughter cells of 
the cambium in the oak, feeling the need of more water-trans- 
porting tissues, would transform themselves into tracheal tubes, 
while in the pine, under like circumstances, tracheids with large 
cavities would be formed. The so-called ring of growth throws 
some light on this question and will now be considered. 

In the stems and roots of trees and shrubs it is found that 
the addition made to the xylem during each growing season 
consists of two more or less well-defined parts, namely, an 
early growth, in which the tracheal tubes are relatively more 
abundant and possess larger cavities (Fig. 24), or where, as 
in conifers, the tracheids have relatively large cavities and thin 
walls ; and a late growth, in which the tracheal tubes are rela- 
tively fewer and smaller, and the tracheids have smaller cavi- 
ties and thicker walls (Fig. 24). In the early growth the 
water-conduction elements may be said to preponderate, and 
in the late growth the strengthening elements; and this is as 
it should be, for the plant first feels the need of water in the 
spring, and later the need of greater strength. The first mani- 
festation of growth in the spring is the unfolding of the 
leaves, and in Dicotyledons and Gymnosperms there are more 
of these than appeared the previous year, for the crown of 
branches grows larger every year; and even if the old tracheal 
tubes or tracheids were in direct communication with the new 
leaves they would not suffice. But the old water channels in 
the stem do not extend into this year's leaves and new ones 



54 



SECONDARY INCREASE IN THICKNESS 



must be formed which will be continuous with those in the 
leaves (see Fig. 25). Later, when most or all of the leaves 

have been formed, and 
tracheal tubes communi- 
cating with these have 
been laid down in the 
stem, the cambium can 
devote itself more exclu- 
sively to the production 
of strengthening tissues, 
the need for which has 
been caused by the in- 
creased size and weight of 
the crown. The two dif- 
ferent regions of a ring of 
annual growth are, there- 
fore, seen to be an ana- 
tomical expression of va- 
rying physiological needs. 
Sometimes it happens 
that trees are stripped of 
their leaves by insects, 
and later become rehabili- 
tated by the growth of 
buds that would under 
normal conditions, have 
lain dormant until the suc- 
ceeding spring. It is then 
found that the new crop 
of leaves stimulates the 
production of a new ring 
of growth in the stem, much as would have happened the fol- 
lowing spring if things had been left to their wonted course. 

In those tropical regions where there is no pronounced dry 
season and the leaves do not all fall off at once, but just a few 
at a time with gradual renewal, there is no ring of growth 




Fig. 25. Diagram showing the relation of 
this year's leaves to the wood of the current 
year. 



GROWTH OF VASCULAR BUNDLES 



55 



formed ; but where in the tropics a decided dry period provides 
plants with too little water, the leaves drop off just as they 
do outside the tropics on the ap- 
proach of winter, and when these 
plants again clothe themselves with 
leaves a ring of growth is formed 
as already described. Such facts as 
these strengthen the conception that 
the formation of the ring of growth 
is at first stimulated by the demand 
for water on the part of the leaves. 
Secondary increase in thickness 
in roots does not differ essentially 
from that of stems, and the slight 
difference that occurs is due to the 
peculiar arrangement of the phloem 
and xylem in the root bundle. It 
will be remembered that the phloem 
and xylem strands in roots stand 
side by side and not in radial line 
as in stems. (Compare diagrams 
in Fig. 21.) When secondary in- 
crease in thickness begins, the cam- 
bium flanking the phloem on the 
inside or toward the center lays 
down xylem elements, so that, with 
the already existing phloem, a col- 
lateral bundle such as is typical in 
stems is produced. At the same 
time the cambium in front of the 
original or primary xylem com- 
monly forms a medullary ray (Fig. 
26), but it sometimes makes phloem 
elements and thus completes a col- 
lateral bundle here also. The cambium then continues to 
add new T phloem and new xylem in both cases, and secondary 




Fig. 26. Cross-section of a 
young root of Phaseolus multi- 
florus. A, pr, cortex; m, pith; 
x, stele (all tissues within the 
endodermis collectively) ; g, g, g, 
g, primary xylem bundle; b, b, 
b, b, primary phloem bundle; 
the cambium, not indicated here 
has the same location as indi- 
cated in C and D, Fig. 21. B, 
cross-section through older por- 
tion of root of the same plant. 
V , b', secondary bast; k, k, 
periderm. The remaining letters 
stand for the same tissues as in 
A. Notice that the cambium has 
laid down medullary rays in 
front of the primary xylem, but 
has made secondary xylem be- 
hind the primary phloem. (After 
Vines.) 



56 SECONDARY INCREASE IN THICKNESS 

medullary rays as the dimensions of the xylem and phloem 
wedges increase, and the root soon comes to look quite like a 
stem, the discernible difference being where the landmarks of 
the primary xylem and phloem can still be made out. 

The purpose of the radial arrangement of the primary xylem 
and phloem of roots appears to be to allow the water absorbed 
by the root hairs to pass into the xylem highways without first 
traversing the food highways in the phloem. The changes 
brought about by secondary thickening would not interfere 
with this purpose because they occur in older parts of the root 
where the root hairs have already died away. 

Increase in the Cortex. — The growth of the vascular bun- 
dles subjects the primary cortex to a good deal of tangential 
tension and its thin-walled parenchyma cells commonly undergo 
enough increase by radial division to keep from breaking 
apart, but increase in thickness of the primary cortex takes 
place in woody perennials and in the underground parts of 
herbaceous perennials by the formation and continued activity 
of a zone of cork cambium or phellogen. This takes its origin 
in the tangential division of the epidermis or in a similar divi- 
sion of the cells immediately beneath the epidermis (com- 
pare Figs. 23 and 24). When the origin is in the epider- 
mis the inner cell cut off by the tangential wall takes part 
in the formation of the cork cambium, while the outer 
cell enlarges and remains epidermal. If the origin is in 
the cell layer just beneath the epidermis it is the outer 
row <of daughter cells that becomes cork cambium. The 
formation of the cork cambium commonly takes place be- 
fore the end of the first year's growth, and by frequent tan- 
gential as well as radial divisions it soon gives rise to layers 
of cork cells toward the outside, and frequently to thin-walled 
parenchyma cells toward the inside called the phelloderm. 
These new tissues, including the cork cambium which forms 
them, are termed the periderm. Since the walls of the cork 
cells are suberized and in uninterrupted union the passage of 
water and gases is prevented. The epidermis being thus shut 



INCREASE IN THE CORTEX 57 

off from the water supply from within soon dies and gradually 
sloughs away ; but provision is made for the aeration of the 
tissues lying within the cork zone by the production of a loose 
mass of cells which interrupts the cork layer and allows the 
air to enter through its intercellular spaces (Fig. 22). This 
aerating tissue is known as a lenticcl. It commonly arises 
beneath a stoma by the division of phellogen cells lying at the 
same depth as those that form the cork, and as it increases in 
size the stoma above it is crowded outward and a rent is made 
in the epidermis. 

In woody plants, as a rule, the cork soon comes to take the 
place of the epidermis, as can be seen by the roughening of the 
surface where the epidermis has disappeared. The phellogen 
first formed near the surface does not remain active indefi- 
nitely and a new one is formed deeper in, which, after a time 
of cork building, is replaced by a still deeper one, and so on. 
Sometimes phellogen layers are formed as deep in as the tis- 
sues of the pericycle, and even within the secondary phloem, 
and large masses of tissues called borke, thus cut off from the 
water supply by cork which the phellogen builds, die, dry up, 
and fall off. This is well seen in the shell-bark hickory, syca- 
more, grape vine, and birch. Frequently the borke clings with 
great tenacity and is furrowed by many clefts as it is stretched 
beyond its strength by the increase in diameter of the stem, 
as seen in the oak, hackberry, and elm. After a time it may come 
about, as the borke falls away, that all of the original primary 
cortex is gone and its place is taken by cork, phellogen, and 
phelloderm; or, to use the collective term, by the periderm; 
and, as has been said, even the pericycle and old phloem tissues 
may be thus replaced. 

The periderm and additions to the phloem by the cambium 
are collectively called the secondary cortex. It will be seen from 
the foregoing that the word cortex has three different applica- 
tions : First there is the primary cortex, extending from the 
epidermis to the pericycle; then there is the secondary cortex 
as just defined ; and finally cortex without qualifying adjective 



58 SECONDARY INCREASE IN THICKNESS 

is applied to all of the tissues outside the cambium ring and is 
synonymous with bark. Borke is a German word meaning 
bark or rind. As frequently used by the German botanists it 
has not the same application as our bark, which includes every- 
thing outside the cambium, but designates those tissues which 
are cut off by the deep-lying cork layers as told above. 

In roots the phellogen takes its origin from the pericycle, 
so that the whole of the primary cortex is shut off from the 
interior water supply as soon as cork is formed, and soon there- 
after dies. It is, therefore, the periderm that constitutes most 
of the bark of old roots. 

Monocotyledons 
Monocotyledons have no cambium ring and additions to the 
vascular bundles can not take place as in Dicotyledons. The 
absence of a cambium ring is due to the fact that the procam- 

bium strands differentiate entirely 
into the permanent tissues of xylem 
and phloem, leaving none of its cells 
in the meristematic condition (Fig. 
28). Increase in thickness in most 
monocotyledons takes place simply by 
the enlargement of the cells of the 
permanent tissues that are formed 
from the primary meristems near 
fig. 27. Photomicrograph the growing apex, and this enlarge- 

of cross-section of very young ment ^ & ml S0Qn ^^ ( compare 

corn-stalk, where the procam- ' v r 

bium strands have just gone FigS. 2J and 28) \ but in palms it COn- 

over into vascular bundles. . r < , . . , . 

For comparison with Fig. 28. tinues for a long time m the ground 

tissue, including the sclerenchyma 
sheath around the vascular bundles, until the diameter of the 
stem has been doubled or trebled. This method of enlarge- 
ment does not increase the number of the vascular bundles, 
nor the capacity of the food and water highways of those 
already existing, and the size of the crown of leaves which 
would evaporate the water and supply the food can not be 




UNUSUAL GROWTH IN THICKNESS 



59 



permitted to increase indefinitely from year to year, as in the 
case of Dicotyledons, where the conducting highways are added 
to each year by the cambium. In palms, for instance, the old 
leaves are shed about as fast as new ones are formed. 



thickness is found 
by the 



in 



the 



genera Dracaena, 




Another method of growth in 
arborescent Liliace?e, represented 
Yucca, and Aloe. Here, 
either close to the growing- 
apex, or remote from it in 
the region of the permanent 
tissues, a secondary meri- 
stem is formed by the tan- 
gential division of the cells 
of the pericycle, which adds 
new cells both inward and 
outward. A part of the cells 
added toward the inside be- 
come differentiated into new 
vascular bundles (Fig. 29), 
and a part into new ground 
meristem ; while those, much 
less in number, formed to- 
ward the outside constitute 
a secondary cortex. In this 
way growth in thickness 
goes on from year to year, 
and has been known to pro- 
duce in Dracaena a stem diameter of fifteen feet. In this 
instance, however, the tree was estimated to be six thousand 
years old. 

Unusual Growth in Thickness. — Variations from the 
usual modes of secondary thickening take place in several fam- 
ilies of Dicotyledons (Apocynacese, Sapindacese, Bignoniacese, 
Chenopodiacese, Amarantacese, Phytolaccacese, Papilionaceae, 
Nyctaginacese), and, under the Gymnosperms, the Cycadacese 
and species of Gnetum. A type of frequent occurrence is 



Fig. 28. Photomicrograph of cross- 
section of corn-stalk somewhat older than 
in Fig. 27. Compare with Fig. 27, and 
notice that the number of vascular bun- 
dles is approximately the same in both, 
and the number of cells in the funda- 
mental tissue is approximately the same. 
Growth in Fig. 28 has been accomplished 
by the enlargement of the cells already 
present in Fig. 27. a, epidermis; b, cor- 
tex and pericycle. 



6o 



SECONDARY INCREASE IN THICKNESS 



where the cambium ring soon ceases its activity and a new ring 
of secondary cambium is formed by tangential division of cells 
of the pericycle, and after this has laid down a zone of vascular 
bundles surrounding those first formed it becomes inactive and 
a new cambium ring giving rise to a new zone of bundles is 
formed outside of it, and so on (Fig. 30, C). A peculiar 




Fig. 29. Portion of a cross-section through the stem of Dracaena marginata. P, 
parenchyma of cortex. V , meristematic zone of the pericycle by the activity of which 
the stem increases in diameter, with the addition of new vascular bundles. M, mature 
vascular bundle. N, nearly mature vascular bundle. 0, newly-formed procambium 
strand from which a vascular bundle is to arise. B, beginning of a procambium 
strand by the division of cells in the meristematic zone. F, parenchyma of the funda- 
mental tissue. (After Haberlandt.) 



type is found in the climbing Serjanias of the family Sapinda- 
ceae where the stem is traversed vertically by several ridges, 
which in cross-section look like lobes, each containing a 
circle of vascular bundles surrounding a pith; and the cen- 
ter of the stem is occupied by a circle of vascular bundles 
in the usual way. The stem is ridged when first formed from 
the primordial meristem, and the primary bundles, following 
the contour of the stem are laid down in the form of a lobed 
circle, as seen in cross-section. When the interfascicular cam- 




Fig. 30. Diagrams showing some 
types of unusual growth in thickness. 
'A, cross-section through a four-year- 
old stem of Anisostichus capreolata; 

B, cross-section of stem of species of 
Bauhinia; the xylem strands, b, are 
stippled while the surrounding paren- 
chyma and bark tissues are left white. 

C, portion of a cross-section of stem 
of Gnetum scandens; 1, 2 and 3 are 
successive rings of growth; m, is the 
pith; b, is a sclerenchyma ring. The 
xylem portions with the exception of 
the larger tracheal tubes are shaded, 
while the medullary rays, phloem and 
tissues intervening between the rings 
of growth and the outer cortex tis- 
sues are left white. (A and C, after 
de Bary; B, after Schleiden.) 



bium is formed it extends 
across the base of each lobe, 
cutting it off from the central 
or main part ; and then it com- 
pletes the circle of bundles in 
each lobe and also the central 
circle, so that each xylem or 
wood cylinder is entire and sur- 
rounded by a phloem cylinder. 

Some of the climbing Big- 
noniaceae vary from the usual 
type by the cambium failing 
to form xylem here and there 
as secondary thickening pro- 
gresses; so that the wood cylin- 
der becomes more or less deeply 
cleft by the phloem (Fig. 30, 
A). In extreme instances the 
wood may become cut up into 
many isolated strands by the 
production of secondary meri- 
stems from cell division in the 
wood parenchyma, medullary 
rays and pith (Fig. 30, B), and 
by ingrowth of cells of the 
pericycle; and the secondary 
meristems may add new tis- 
sues to these strands and even 
interpolate new strands amongst 
them. 

The growth in thickness of 
fleshy roots, tubers, and rhi- 
zomes, does not, as a rule, dif- 
fer in method from the normal 
type, but the cambium gives 
rise principally to wood paren- 
chyma and medullary ray cells, 
or the cells of the primary cor- 



62 SECONDARY INCREASE IN THICKNESS 

tex and pericycle multiply enormously and constitute most of 
the thickening ; or, again, the secondary cortex may be chiefly 
involved, as in the root of Taraxacum officinale. A notable 
exception to the normal type is found in the root of the beet 
where successive secondary cambium rings are formed outside 
the first and by their activity bring about the thickening of the 
root. 

Illustrative Studies 

i. Make cross and longitudinal sections of a stem of Aris- 
tolochia that is several years old (Fig. 24). The sectioning 
will need to be done on a sliding microtome, and the sections 
should be stained with erythrosin and iodine green, or with 
safranin and hematoxylin, and made into permanent mounts 
in balsam. Study with low and high powers "and note the 
changes which each tissue has undergone since the last condi- 
tion studied. What tissues have increased? decreased? been 
broken ? crushed ? Have new tissues appeared ? Make draw- 
ings and diagrams to show the changes discovered. 

2. Pay particular attention to the new medullary rays. 
What evidence can you find as to how they have originated? 
Assuming that the function of the rays is to carry water and 
food radially and to store them also, can you see special need 
of the new rays? 

3. Find the primary xylem and phloem (page 37) of the 
vascular bundles. Have they been changed in position and 
condition since first they were formed from the procambium ? 

4. Study cross-sections of corn stalk close to the growing 
apex and farther down where growth in diameter is well 
marked. As the stem increases in diameter do the vascular 
bundles enlarge ? Does the number of cells in them increase ? 
Does the number of vascular bundles increase? What 
changes take place in the ground tissue as stem enlargement 
progresses? Do you find cambium in the vascular bundles? 
Draw a vascular bundle on a large scale and show how it dif- 
fers from a dicotyledonous bundle (of Aristolochia, for in- 
stance). 



ILLUSTRATIVE STUDIES 



C>3 



Good material for this study of corn can be obtained from 
stalks of field corn about two feet high. By stripping away 
the leaves and leaf sheaths the stem will be found extending 
about six inches up from the ground with nodes and inter- 
nodes in all stages of development. 



* 



CHAPTER IV 

PROTECTION FROM INJURIES AND LOSS OF WATER 

The simplest unicellular plants cover themselves with a thin 
cellulose cell wall. This is harder and tougher than the proto- 
plast and preserves its form and protects it from injuries ; but 
since it must be of a nature to allow the passage inward of 
water and solutes it can not be efficient in keeping the proto- 
plast from drying up. It is found that if a cell wall will allow 
water to pass in it will also let it out ; and while the plasmatic 
membrane is able to retain osmotic substances in solution in 
the water of the cell sap it is unable to prevent the loss of the 
water itself. The unicellular plants, or those consisting of a 
few cells only, have found that they can inhabit only wet and 
moist places, or else be able to exist for longer or shorter 
periods in a condition of extreme desiccation. As soon as 
plants in the course of their evolution increased in size and 
complexity and began to burrow into the earth for its treasures 
of water and minerals, and, at the same time, to rise into the 
air and light, where they could appropriate other raw materials 
and the sun's energy, they found themselves exposed to danger 
of destruction from mechanical injuries and loss of water ; and 
they found in working out the problem, which such plants 
have had to solve, of division of labor amongst different sets 
of cells composing the body, that it was necessary to assign 
one or more exterior tissues to the function of protection. 

Two tissues, the epidermis and the cork, have accordingly 
been evolved for the specific purpose of protection, and two 
others, the collenchyma and superficial sclerenchyma cells and 
fibers which belong to the skeleton of the plant, also give pro- 
tection by reason of their hardness, toughness, and tensile 
strength. Without such safeguards large terrestrial plants 

64 



I rll'IKMIS AS I'ROTECTIYK 1'ISSl'l-: 



65 




B «QO_Or 



standing- ready at any time to make use of available materials 
and forces never could have been wrought out. 

The Epidermis 
The Epidermis as a Protective Tissue. — The epidermis is 

the first, and often the only, protective tissue formed, and 
since it lies at the exterior it must bear the brunt of adverse 
conditions in the environment. It prepares itself for its task 
mainly by modifications of its outer wall, which it thickens 
and waterproofs according to the 
demands made upon it. The thick- 
ness of an average outer epidermal 
wall under normal conditions of 
moisture in the substratum and sur- 
rounding atmosphere is not far from 
.0033 mm., or about one thirty- 
sixth the thickness of this page; but 
as greater dryness in the environ- 
ment imposes severer conditions, 
and where the epidermis persists 
through several years with incident 
wear and tear, we find the outer wall 
often increasing in thickness, even 
up to .03 mm., or one fourth the 
thickness of 'this page, as in the 
case of the mistletoe (Fig. 31). 
Measured by ordinary standards 
the epidermal outer wall is in any 
case extremely thin and would seem 
poorly adapted to withstand any 
kind of mechanical attack; but it 
must be remembered that the epider- 
mal cells are very minute, averaging about .03 mm. in tangen- 
tial diameter, so that the radial walls serving as a foundation 
on which the outer wall rests, are that distance apart merely. 
Any stress upon the outer wall is, therefore, not borne by it 






o 



Fig. 3.1. A, cross-section 
through upper half of leaf of 
Pyrus Japonica, showing cutin- 
ized layer of the outer wall at 
f, and cellulose layer at g. B, 
the same for the leaf of Rus- 
sian olive; the cutinized layer 
is thinner and the cellulose 
layer thicker than in the former 
instance. C, portion of cross- 
section of submerged stem of 
Nymphsea odorata, where there 
is no cutinized layer, and the 
cuticle is a hardly-distinguish- 
able film. 



66 PROTECTION FROM INJURIES AND LOSS OF WATER 

alone, but by the radial walls also and the underlying tissues 
with which these are connected. Added to this the outer wall 
commonly curves outward, as it spans the space between the 
radial walls (Fig. 14) and is thus made more resistant against 
stress from without. The part which the radial walls play as 
props for the outer wall will be better apprehended by an 
example. Assuming that the tangential diameter of the epi- 
dermal cells is .03 mm., and that the cells as seen from the 
surface are four-sided, then if any object 1 sq. mm. in cross- 
section were pressing upon the epidermis there would be more 
than 2,000 radial walls immediately beneath and sustaining 
that portion of the outer wall upon which the pressure comes. 
Of course the smaller the object which is pressing upon 
the surface the better chance it has of piercing the outer 
wall. Since the point of an ordinary needle would cover not 
more than one quarter of the surface of our average epidermal 
cell it could be placed so as to avoid the radial walls in piercing 
the surface. Experimenting on leaves grown under average 
conditions it has been found that when using such a needle a 
pressure of .55 gm. to .8 gm. was necessary to break through 
the outer wall of the upper epidermis. 

Incrustations and infiltrations of silica and calcium carbon- 
ate often contribute to the defense of the outer wall, and this 
to a very notable extent in Equisetum and many grasses where 
silica is very abundant. 

The cutinization of the outer wall, which is primarily for 
waterproofing, increases the power to resist tearing, experi- 
ments having shown that the cutinized wall may be even ten 
times as strong in this respect as ordinary cellulose walls. 

Turning from the anatomical and experimental details we 
find nature giving a broad and convincing answer regarding 
the mechanical efficiency of the epidermis. Leaves, on the 
whole, outride the storms and vicissitudes of three seasons 
practically uninjured; and herbaceous plants run their course 
with nothing but the epidermis to cover them. In the whole 
group of fleshy fruits the epidermis has been found an ade- 



EPIDERMIS AS WATERPROOF COVERING 67 

quate protection until the time of ripening; and in many woody 
plants, such as species of Acer, Rosa, Cornus, Acacia and Cin- 
namomum, the epidermis, continually renewing the outer wall 
as it wears away, remains as the sole outer covering for many 
years. 

The epidermis is, however, not proof against all attacks that 
plants are subject to; insects seem to have little difficulty in 
gnawing through or piercing the outer wall, and many para- 
sites excrete ferments that render it soluble, and storms some- 
times overtax its strength. But such things are on the whole 
not sufficiently severe and widespread, at least in the course of 
a single growing season, to make a serious demand for reen- 
forcement of the epidermis. 

The Epidermis as a Waterproof Covering. — The chief 
value of the epidermis lies in the protection which it gives 
against a too rapid evaporation of water. All terrestrial plants 
of any size would lose water faster than they can absorb it 
but for the protection which the epidermis affords. As stated 
in the previous chapter, the waterproofing of the epidermis lies 
in the cuticle and cutinized layer of the outer wall. The cutin 
which gives these their peculiar character is waxy in its nature, 
and when present in abundance it makes the wall practically 
impervious to water. The cuticle seems to be nearly pure 
cutin, while the cutinized layer appears to contain a certain 
percentage of unaltered cellulose. In many cases the cutinized 
layer is absent and then the outer wall consists of cellulose 
bounded externally by the waterproof cuticle. We adopt the 
scheme of plants when w r e pour paraffin over jelly to keep it 
from drying out and moulding, and when we coat paper with 
paraffin for waterproofing purposes. 

The efficiency of the epidermis in preventing loss of water 
is seen by comparing the amount of loss where the epidermis 
is removed in some cases and left intact in others. For in- 
stance, two apples were hung up in a dry atmosphere, one pared 
and the other uninjured, and after 48 hours the former had 
lost 33 per cent, of its original weight and the latter 1 per cent. 



68 PROTECTION FROM INJURIES AND LOSS OF WATER 

An Aloe leaf, according to Haberlandt, had in 24 hours lost 
15.6 times more water where the epidermis was removed than 
where it was left on. 

As might be expected, the amount of cutinization, as well 
as thickness of the outer wall, depends upon the severity of the 
demands made by the environment. In submerged water 
plants the outer wall is usually thin and little if at all cutinized, 
while parts rising above the surface of the water show greater 
thickness of wall and more cutinization. In land plants the 
waterproofing characters become more pronounced and reach 
their fullest development in desert regions, or in alpine and 
arctic regions, and bogs and salt marshes, where the water, 
although present in abundance, is difficult of absorption on 
account of its low temperature or the inimical nature of the 
substances dissolved in it. Plants of the same kind grown in 





D 



^=tH^ 







G^ccca 

Fig. 32. A, portion of cross-section of leaf of Avicennia growing in salty soil; 
outer wall of epidermis very thick. B, cross-section through skin of apple. C, cross- 
section through upper half of petal of Japan quince. D, upper, and E, lower epidermis 
of leaf of Hibiscus moscheutos. F, epidermis of leaf of Lactuca scariola in the sun; 
and G, in the shade. 

dry and in moist atmospheres are apt to differ decidedly in 
their epidermal defenses; and even the different parts of the 
same plant or the same members of a plant are apt to differ 
in this respect according to the demands made upon them. 
Thus, the upper epidermis of a leaf has, as a rule, a thicker and 



CELL CONTENTS OF EPIDERMIS 69 

more highly cutinized outer wall than the lower epidermis 
(Fig. 32), and the epidermis of the petals of a flower, which 
are to endure for so short a time, is insignificant in its defensive 
characters in comparison with the epidermis of the fruit which 
is to last through a much longer period and endure greater 
hardships (Fig. 32). 

The Radial and Inner Walls of the Epidermis. — The 
radial and inner walls are usually thinner than the outer. 
Cutinization sometimes extends for some distance into the 
radial walls, but it seldom involves the whole of the radial wall 
or any part of the inner wall. It has already been stated that 
the epidermis remains alive so long as it is not cut off from 
water by the formation of cork beneath it, and the relative 
thinness and non-cutinization of the radial and inner walls per- 
mit the inflow of water and the interchange of materials neces- 
sary to all living cells. 

The Cell Contents of the Epidermis. — The cell cavity is 
usually quite clear and is evidently serving as a reservoir for 
surplus water. The protoplast lines the wall as a very thin 
film, and although so well exposed to the light it seldom con- 
tains chloroplasts excepting in the guard cells of the stomata, 
and in the case of some plants, particularly Monocotyledons, 
of shady habitats. Leucoplasts are frequently present, and in 
flowers and fruits they often become transformed into chromo- 
plasts and produce the yellow, orange, and some of the red 
colors. Not infrequently blue, violet, and some qualities of 
red pigments occur in solution in the sap of the epidermal cells 
of flowers and fruits, young leaves in the spring, the upper 
epidermis of some alpine and tropical plants where absorption 
of a part of the intense sunlight before it reaches the chloro- 
phyll apparatus may be of use, and of the lower epidermis of 
some shade-loving plants where it may be of advantage in 
absorbing more of the sun's energy before it escapes at the 
lower surface. We assume that the pigments in fruits and 
flowers attract insects and other animals that may be of use in 
pollination and in dissemination of seeds. The use of pig- 



70 PROTECTION FROM INJURIES AND LOSS OF WATER 



ments in the cell sap of young leaves seems to be to protect 
the chlorophyll against the destructive chemical changes which 
are induced by strong light. In addition to pigments tannins 
not infrequently occur in the epidermal cells, where they may 
be of use in warding off attacks of animals and fungous 
parasites. 

Outgrowths and Excretions of the Epidermis. — Excre- 
tions of wax in the form of rods, scales, and grains often 
occur over the epidermis of fruits, leaves, and tender stems 

(Fig. 33). These, as experiments 
show, materially assist the water- 
proofed outer wall in preventing loss 
of water. 

Outgrowths of the epidermal cells 
in the forms of hairs, both slender 
and spinous, and scales, are of fre- 
quent occurrence. In forming these 
the epidermal cells may grow out- 
ward without undergoing cell divi- 
sion, or they may undergo cell divi- 
sion and give rise to multicellular 
protuberances. These are apt to vary 
in different species in size, form, 
complexity, and other characteris- 
tics (Fig. 33), and are very use- 
ful to the microscopist in detecting 
the source and purity of powdered 
foods and drugs. Although in many 
instances these structures appear to 
be mere caprices of growth without 
any function assigned to them, yet, in other cases, their use is 
very apparent. They are, indeed, employed in a wide range of 
service, being used as a protection against injury, to assist 
climbing plants in holding on to their support, in the scattering 
of fruits, in the reduction of transpiration and the intensity of 
illumination, in the secretion of special substances ; and in the 
absorption of water and other materials. 




Fig. 33. Different forms of 
epidermal outgrowths. 1, hooked 
hair from Phaseolus multiflorus; 
2, climbing-hair from stem of 
Humulus Lupulus; 3, rodlike 
wax coating from the stem of 
Saccharum officinarum; 4, climb- 
ing-hair of Loasa hispida; 5, 
stinging hair of Urtica urens. 
(Fig. 3 after de Bary; the re- 
mainder from Haberlandt.) 



THE MULTIPLE EPIDERMIS /I 

All of those hairs and scales that are more or less rigid and 
rough, sharp-pointed or barbed, offer difficulties to browsing- 
animals that would tend to lessen their onslaughts. In the 
stinging hairs of the Urticas the device for protection has 
readied a high degree of efficiency (Fig. 33). Here the outer 
wall is silicified about the apex, and is so thin and brittle that 
it breaks on slight pressure and the jagged edges of the frac- 
ture prick through the skin, while a stinging fluid is injected 
into the wound by the pressure that breaks off the point, and 
doubtless also by the elasticity of the turgid cell. In Humulus 
Lupulus, Phaseolus multiflorus, and Loasa hispida we find 
excellent examples of hairs that act as hooks to assist plants 
in climbing (Fig. 33), while in the branched and interlaced 
hairs of Verbascum Thapsus, and the stellate scales of species 
of Abutilon, Olea and Croton are efficient devices for reducing 
transpiration and reflecting a part of the sun's rays. The 
reduction of transpiration is brought about largely by the for- 
mation of dead air spaces between the interlaced hairs and 
beneath the scales. 

Hairs that are used for absorption and the secretion of 
special materials will be discussed in the chapters on absorp- 
tion and secretion. 

The Multiple Epidermis. — Sometimes the outer layer of 
epidermal cells is underlaid by one or more layers similar to it 
in content. These cells may have arisen from a division of 
the protoderm, in which case they are morphologically or by 
descent epidermal, or they may have sprung from the ground 
meristem beneath the protoderm, and then they would be not 
strictly epidermal, although so classed by general usage. 
Whatever their origin, these cells, together with those of the 
outer layer, are collectively called the multiple epidermis (Fig. 
13). The inner or accessory cell layers as a rule seem to serve 
almost exclusively as water reservoirs. In most cases their 
walls remain cellulose and thin and their cavities become rela- 
tively large. As in the outer layer, the protoplast is a thin film 
lining the w 7 alls, and the cell cavity is filled with a clear fluid, of 



72 PROTECTION FROM INJURIES AND LOSS OF WATER 

course mostly water. Since these accessory layers are chiefly 
for holding water they should be discussed in the chapter on 
storage of reserve materials, but they also have a place here 
because they protect the chemically active cells beneath by filter- 
ing out some of the sun's heat 

— qoOqo^ooc and by ^pp 1 ^ them with 

^v"^ — YAf~\i lT"~Y water when evaporation is reduc- 
^^rf ^jiV^^f m S tn ei r turgidity too low for the 

• = — = ^^==^^j^^r- y= normal performance of their 

functions. In some cases, how- 

Fig. 34. Multiple epidermis of 

leaf of mangrove in cross-section, ever, the accessory layers have 

This serves as a water reservoir thick waUg and re l at i ve l y sma U 
and the relatively thick walls of J 

the inner ceils reenforce the pro- cavities and assist the outer layer 

tective power of the outer layer. t . n . 

The mangrove grows in the salt soil chiefly in protecting against me- 
of sea-coasts. chanical injuries (Fig. 34). 

The Cork 

It is the rule in the perennial parts of trees and shrubs that 
the epidermis is sooner or later replaced by the cork tissue 
(Fig. 24) ; in many instances the change beginning in the first 
year, and in others not until the lapse of many years. The 
cork then assumes the protecting and waterproofing functions 
of the displaced tissue. 

By additions from the phellogen or cork cambium the cork 
tissue becomes several to many cell layers thick. After the 
cork cells have attained their growth they die and their cell 
sap is replaced by air, a fact which accounts for the lightness 
of cork. The walls of the cork as a rule are thin and in some 
instances suberized through and through, while in others a 
middle lamella of cellulose is present. The suberization or 
waterproofing of the wall is accomplished by chemical changes 
in the original cellulose wall and by additions of suberin layers 
to this, or by the latter process alone. The suberin is quite 
similar to cutin in its chemical constitution and physical prop- 
erties, and both are of the nature of wax. 

Where the cork becomes only a few cell layers in thickness 



CORK AS WATERPROOF COVERING 73 

the cells are apt to be flattened so that the tangential diameters 
are broader than the radial, but where annual additions are 
made by the phellogen, the cells first formed are not so flat- 
tened and may be even larger in the radial diameter, and these 
are succeeded at the close of the season's growth by radially 
narrower cells, so that rings of growth appear in the cork as 
well as in the wood. This is seen in bottle cork as alternating 
light and dark bands. 

Cork as a Protectiye Tissue. — The fact that the cork 
tissue is several cell layers in thickness makes it better than the 
epidermis as a buffer against stresses from without ; and since 
in perennial parts the phellogen may keep up its activity from 
year to year any injuries and losses to the cork are quickly 
repaired, and therefore the cork is, on the whole, better than 
the epidermis where chances of injury are multiplied by years. 

The many suberized walls of the cork tissue interpose a 
series of barriers against the ingress of fungal parasites. The 
sweet and Irish potatoes afford examples of the effectiveness 
of cork in this respect. Here the cork is quite thin, averaging 
hardly more than six layers of cells in thickness, and yet the 
potatoes remain sound until the cork covering is broken 
through, when decay is apt to set in very quickly, because the 
omnipresent microscopic bacteria and spores of fungi can now 
get at the deeper and less resistant tissues. 

Cork also affords protection from danger of another kind; 
air is a very poor conductor of heat, and each cork cell em- 
braces a dead air space which prevents sudden interchanges of 
temperature. If the outside temperature suddenly falls its 
effect can not at once be felt t>v the deeper tissues, and onlv 
little by little can the heat of the plant be dissipated through 
its cork jacket. Again, after the plant has once frozen, if the 
exterior temperature suddenly rises, the cork may prevent death 
that is so apt to result to the tissues from sudden thawing. 

The Cork as a Waterproof Covering. — The walls of the 
cork cells are permeable with great difficulty to water and 
gases. Experiments with the Irish potato have shown that 



74 PROTECTION FROM INJURIES AND LOSS OF WATER 



in 48 hours a potato with the cork covering* removed lost sixty 
times as much water as an unpeeled one of equal weight. It 
was found by Wiesner that a film of cork two or three cells 
in thickness allowed no air to pass through it, even under a 
pressure of more than a third of an atmosphere continued for 

several weeks. This gives us an 
exact statement of a fact that every 
one apprehends in general terms 
from our experience with cork stop- 
pers, namely that fluids in a state of 
vapor are practically unable to pene- 
trate them. 

Use of Cork in Healing 
Wounds. — In case of injury to 
stems and roots, as when the bark 
is gnawed or branches are broken 
off by storms or pruned away, the 
parenchyma cells of the cortex and 
pericycle in the region of the wound 
form secondary meristems by cell 
division, which build the various 
tissues of the bark until the wound 
is closed over and form a new cam- 
bium layer where that has been torn 
away, and a phellogen which gen- 
erates an exterior covering of cork. 
fig. 35. Diagram illustrating When leaves ripen and fall away the 

the relative development of the t1 1 . r 1 j 

protective tissues in different parts Cells at the Surface Of the WOUnd 

of a plant. Description in the become suberized and are in effect 

text. 

cork cells. 
The relative dependence of the different parts of a plant on 
epidermis and cork is shown diagrammatically in Fig. 35. 
The fact that a waterproofed epidermis does not occur at the 
growing apex is indicated by a very thin line. As the epi- 
dermis becomes better developed on the successively older 
leaves and portions of stem and root the line is thickened. 




OTHER MEANS OF PROTECTION 75 

The evanescent flower does not demand as effective an epi- 
dermis as the leaves and stem, and this is here indicated by its 
thin outline. The pistil persists, and as it develops into the 
fruit it perfects its epidermis as a waterproof covering, as 
indicated by the thick outline of the fruit in the diagram. 
Finally on the older portions of stem and root cork appears 
(the barred zone in the diagram) and gradually increases until 
it bursts the epidermis, and after a time takes its place 
altogether. 

Other Means of Protection 

Since the epidermis and cork lie at the very exterior they 
may be classed as preeminently protective tissues; but other 
tissues lying near the surface may be of a nature to give the 
cork and epidermis substantial assistance; and although the 
chief function of these may lie in another direction they can 
not be passed unmentioned when the function of protection is 
being discussed. The collenchyma and sclerenchyma tissues 
belong in this class. 

The origin and general character of the collenchyma has 
been told on page 33. It forms a somewhat rigid foundation 
for the epidermis and cork of superficial origin. As stated 
on page 33, the term sclerenchyma is applied to those cells of 
whatever form whose walls are on the whole uniformly thick- 
ened and commonly lignified. When short and stocky these 
are called stone cells, and when long and slender they are 
termed bast fibers. These not infrequently occur close to or 
just beneath the epidermis or periderm and help to keep these 
from caving in, and breaking under the stress of a blow or 
pressure from without. In stone fruits, such as the walnut 
and peach, it is the stone cells forming the stone that afford 
the sole means of protection to the seed after the pulp or shell 
has been removed. 

The borke (p. 57), although a mass of dead tissues in 
process of elimination, is of no small use as a protective cover- 
ing; indeed, the very fact that it is dead and dry and hard 



7& PROTECTION FROM INJURIES AND LOSS OF WATER 

gives it its protective value. Good illustrations of this are 
found in the shellbark hickory, sycamore, and grapevine. 

Illustrative Studies 

i. Enclose a bit of apple peeling in elder pith (page 218) and 
cut free-hand sections. Mount some of the thinnest of these 
in a drop of dilute glycerine. Draw to scale a few of the 
epidermal cells. Measure the cell cavity and the thicknesses 
of the walls. 

Note the color of the walls in dilute glycerine and mount 
other sections directly in chloroiodide of zinc. The walls that 
turn yellow in this are cutinized and those that turn blue are 
cellulose. 

2. Select two similar apples and pare one. Weigh them 
both and hang them up by their stems. After forty-eight 
hours weigh them again and estimate the percentage of loss 
of water in each case. 

3. Heat a glass slide quite hot and press the skin of an 
apple against it. Notice the waxen spot that is left on the 
glass. 

4. Study cross-sections of a marshmallow leaf, for instance, 
and measure the thickness of the outer wall of the upper and 
lower epidermis. Draw a few cells from each epidermis to 
scale. 

5. Make note of what becomes of the epidermis in the stem 
of Aristolochia as the stem grows older, using the permanent 
mounts already prepared for the previous chapter. 

6. Strip the epidermis from an Iris leaf or any other leaf 
from which the epidermis is readily removed and mount it in 
a drop of dilute glycerine. Count the number of radial walls 
(walls on edge in the preparation) in one or more squares of 
the eyepiece scale and estimate the number of these in a square 
millimeter. 

7. Determine from cross-sections of old and young Aris- 
tolochia stems where the cork in the old stems comes from. 
Measure an average cork cell and the thickness of its walls. 
Draw a few cells to scale. 



ILLUSTRATIVE STUDIES 77 

Mount a cross-section of old Aristolochia stem in chloro- 
iodide of zinc, and the walls of the cork cells should turn 
yellow. Measure the thickness of the entire cork layer. 

8. Cut in elder pith sections from a bit of potato peeling 
that has been hardened by standing in 95 per cent, alcohol. 
Study sections in dilute glycerine and chloroiodide of zinc. 
Compare the cork cells found here with those in Aristolochia. 
Measure the thickness of the cork layer. 

9. Select two potatoes of about equal size and pare one. 
Weigh them and hang them up for forty-eight hours; then 
weigh them again and estimate the percentage of loss of water 
in each case. Compare these figures with those from the 
apple. Does either tissue (cork or epidermis) seem to be 
more efficient than the other? 

10. Study cross-sections of leaves of mullein, Russian olive, 
Crotonopsis, or sections of other leaves having a dense coating 
of hairs and scales. 

Make drawings to show how these trichomes (outgrowths 
from the epidermis) can help in reducing illumination and 
transpiration. 

11. Cut out a piece of the hollow stem of Equisetum, and, 
beginning with the inner surface, scrape away the tissues down 
to the silicious outer incrustation of the epidermis. Put this 
preparation into a strong chromic acid solution to macerate 
and separate the organic parts from the incrustation. Rinse 
the preparation and at the same time brush it with a soft brush. 
Mount the preparation in a drop of dilute glycerine and study 
it with low and high powers. Note how complete is the in- 
crustation, and the details about the stomata. Make a draw- 
ing to show these things. 



CHAPTER V 
THE PLANT SKELETON 

The cell wall of unicellular plants such as Pleurococcus and 
yeast is essentially an exoskeleton since it provides some degree 
of strength and rigidity, and this is true of the cell walls in 
the higher plants ; but where a plant on account of its size and 
exposure to the elements is in danger of breaking down or 
being crushed or torn it has been found necessary to set apart 
certain tissues as a skeleton for the plant body as a whole, and 
these tissues have been modified to become more effective as 
skeletons . and at the same time less efficient for other func- 
tions. The need of a skeleton for the larger and more com- 
plex plants is at once apparent. The larger the plant the 
greater is its tendency to collapse on account of its own weight. 
Imagine a tree trying to attain its normal size with all of its 
tissues like elder pith or the pulp of an apple, or a toadstool 
presuming to become the size of a tree without making any 
tissues like bast or wood ! The more branched a plant is the 
greater is its danger of becoming dismembered, and the 
greater is the need of the body being strong to support the 
branches, and of the branches being firmly knit to the body. 
The more differentiated the plant body becomes the greater is 
the danger attending dismemberment ; and the greater also are 
the demands made on the environment; and the catastrophe 
is correspondingly more serious when any parts are torn away 
or thrown out of their normal positions. Hence it is that the 
higher plants have been under the necessity of building a skel- 
eton of greater or less strength and hardness. 

We find that for the purpose of a skeleton four tissues have 
been wrought out, whose origins have already been told in 
Chapter II, namely the collenchyma, the bast fiber tissue, the 
wood fiber tissue, and the stone cell or stereid tissue. 

78 



THE COLLENCHYMA 79 

The Making of the Skeleton. — The necessary strength and 
hardness of the skeleton is obtained by modifications of the 
cell wall and changes in the forms of the cells. The ordinarily 
thin walls do not suffice to hold the plant body erect by their 
own strength, but when filled with cell sap until they are 
stretched they become rigid, like a toy rubber balloon when 
inflated with gas, and in that way hold the body firm and erect. 
Plants or parts of plants that depend upon this condition for 
strength soon wilt when they begin to lose water faster than 
they take it in. 

The skeletal tissues make plants more or less independent 
of fluctuations in the water supply in maintaing the right 
form and position of the body. The modifications of the wall 
involve thickening of the originally thin wall, chemical altera- 
tions of the wall by changes in the old materials and deposi- 
tions of new, and transformations in the physical condition 
of the wall, such as its hardness and elasticity. The changes 
in the form of the cells consist, as a rule, in their elongation 
parallel with the line of- action of the main forces which they 
are to resist. While the cells are elongating their ends usually 
glide past each other and form dovetailed ioints which greatly 
increase the strength of the tissue (Figs. 15 and 19). 

The Tissues of the Skeleton 
The Collenchyma. — This is the first skeletal tissue formed. 
It appears in stems a short distance below the growing apex 
where the bast and wood have as yet not begun to be formed, 
and it is therefore the only tissue thus far having a strength- 
ening function chiefly. Its chief characteristic is that its walls 
are thickened at the angles where three or four cells join, and 
this thickening in extreme cases becomes so great as to almost 
close the cell cavity. While the angles are thickening a median 
strip of the wall is left thin, clearly in order to allow a flow 
of sap from cell to cell. The walls, as a rule, remain cellulose 
throughout (Figs. 11 and 14). 

Since the collenchyma is formed where growth in length of 






SO THE PLANT SKELETON 

the stem is still taking place it must be capable of growing 
itself or of stretching and offering a moderate resistance only 
to the increase in size of the other tissues. For this reason it 
can not be relied on as the mainstay very far down the stem 
where the stresses to be overcome are greater than in the 
region of the apex, and the bast fiber tissues are formed there 
to reenforce it (Fig. n). The elastic strength of the collen- 
chyma is small compared with that of wood and bast fibers, 
and when the elongation of the stem is rapid it is continually 
stretched beyond its limit of elasticity. This is shown by the 
fact that such stems on wilting droop through several inter- 
nodes dominated by the collenchyma. We may therefore look 
upon the collenchyma as a compromise between the need for 
strength and the need to elongate during growth in length. 

When growth in thickness sets in the collenchyma becomes 
stretched tangentially and may even be broken apart in many 
places (compare Figs. 23 and 24) ; but this is not apt to take 
place until the bast fibers or the wood fibers also are ready to 
reenforce it. Through all these vicissitudes of stretching and 
tearing the collenchyma remains alive, till the end of the sea- 
son in annuals, and in perennials till the formation of a deep- 
lying cork tissue shuts it off from the supply of water and sap. 

In a very subordinate way the collenchyma may be used for 
the slow conduction and temporary storage of materials, and 
since it sometimes contains chloroplasts it may take part in 
the manufacture of food (see Chapter IX). 

The Bast Fibers. — As has been learned in Chapter II, the 
bast fiber tissue may occur in the primary cortex, pericycle, or 
secondary cortex. Where it occurs as a primary tissue in the 
primary cortex or pericycle it is formed below the apex where 
growth in length has ceased, following next to the collen- 
chyma in time (Fig. 11). It may occur anywhere in the 
regions named, from immediately beneath the epidermis to a 
position in front of and in contact with the phloem portions 
of the vascular bundles. It may be in isolated longitudinal 
strands or in the form of an unbroken hollow cylinder (Fig. 17). 



THE BAST FIBERS 8 1 

The walls of the bast fibers become much thickened, even in 
extreme cases to the entire closing of the cell cavity. The 
length of the fibers varies within wide limits : in some cases 
they are no more than I mm. long, while in flax they reach 
a length of 40 mm. and in hemp jj mm. The average length 
is about 2 mm. It will readily be seen by these figures how 
it is that the fibers of flax and hemp can be twisted into thread 
and woven into cloth, while those of most plants are much too 
short for the purpose. The length of the fibers has much to 
do with the strength of the bast tissue as a whole, for, as has 
already been said, as the nascent fibers elongate their ends 
glide by one another, and the length of the splice increases 
with the length of the fibers. As a rule the walls of the bast 
fibers become decidedly lignified, but all grades of condition 
occur from almost pure cellulose to complete lignification. 

In those herbaceous plants whose cambium produces little or 
no wood fiber tissue the bast remains the chief dependence for 
strength; but where the wood is much represented as in the 
older parts of many annual stems and in all woody perennials, 
the significance of the bast as a strengthening tissue falls into 
the background. This fact is quickly appreciated by noticing 
how little in such cases the strength of the stem is diminished 
by stripping off the bark. 

The bast fibers are employed not only to strengthen the stem 
as a whole, but also to protect and give stability to the delicate 
tissues of the primary and secondary phloem, as when the bast 
strands stand like a buttress before the primary phloem or, in 
the secondary phloem, are built by the cambium alternately 
with groups of sieve tubes, companion, and parenchyma cells. 
In Monocotyledons a fibrous tissue similar to the bast of 
Dicotyledons surrounds each isolated vascular bundle more or 
less completely (Fig. 40). 

The bast fibers are specially fitted for their mechanical func- 
tion by their great elastic strength. (By elastic strength is 
meant the measure of the force required to stretch the fibers 
to the point beyond which they are unable to return to their 
7 



82 THE PLANT SKELETON 

original length.) In this respect they are quite equal to 
wrought iron, and in some instances they equal and even sur- 
pass steel. But they surpass both iron and steel in a respect 
of great importance to plants : they are able to stretch many 
times as much as the former before the limit of elasticity is 
reached. This quality enables plants to bend before the wind 
and spring back to their original positions when the stress 
is past. 

When the bast fibers have reached their full development 
they die and their cell cavities become filled with water or air, 
but they continue none the less effective as skeletal tissues. 

The lengths and frequency of occurrence of the bast fibers 
often afford good evidence in the detection of the purity of 
powdered drugs. 

The Wood Fibers. — The wood fibers are those bastlike 
fibers that occur in the wood or xylem portions of stems and 
roots. In most cases they are the product of the cambium 
alone, and not of the procambium, and we therefore expect to 
find them in the secondary, but not in the primary xylem. 
While they are similar to the bast fibers in being elongated 
and tapering and having thickened and lignified walls, they do 
not equal the bast in length. They are rarely as long as 1.5 
mm. and are known to be as short as o.? mm. Since, as a 
rule, they are derived from the cambium only, they do not 
occur as close to the growing apex as do the bast fibers, but 
farther back where secondary increase in thickness has begun 
(Fig. 11). 

The proportion of wood fibers to the other elements of the 
secondary xylem varies greatly in different species. In many 
woody Dicotyledons the wood fibers constitute the greater part 
of the secondary xylem or wood; but even in this class of 
plants cases occur where the other elements of the xylem 
greatly preponderate. 

The specific gravity, hardness, elasticity, and strength of the 
wood depend upon many factors, not all of which are referable 
to the wood fibers themselves. The proportion of wood fibers 



THE WOOD FIBERS 



83 



and the thickness and physical character of their walls are all 

important factors in the quality of the wood; but the plan of 
distribution oi the tracheal and thin-walled parenchyma ele- 
ments causes the libers to occur in larger or smaller groups in 
the different species, thus making the wood as a whole stronger 
or weaker in consequence. 




Fig. 36. Photomicrograph of cross-section of oak wood. E, early growth; L, late 
growth; m, larger medullar y ray; n, smaller ray. 



In woody plants the wood fibers are, as a rule, relatively 
much more numerous in the late than in the early growths. 
This difference stands out sharply in such woods as the ash 
and the oak (Fig. 36) where the late growth is a dense, hard, 
and strong cylinder encasing' the relatively weak and porous 
cylinder of the early growth. In some trees, like the yellow 
poplar of commerce, the wood fibers are relatively few through- 
out the entire year's growth, and the wood is relatively light, 
porous and weak in consequence (Fig. 37). Although the 
quality of the wood depends much upon its visible characters, 



8 4 



THE PLANT SKELETON 



such as the frequency of its wood fibers and the thickness of 
their walls, other invisible characters, such as the hardness, 
elasticity and breaking strength of the walls are factors so 
important that whether a wood should be classed as hard or 




Fig. 37. Cross-section of yellow poplar wood. E, early; L, late growth; m, medullary 
ray. Photomicrograph. 



soft, weak or strong, could not be determined by microscopic 
examination alone. 

Sometimes the wood fibers are entirely lacking, as in Aris- 
tolochia sipho, where their place is taken by tracheids, and as 
in pine and other Gymnosperms where tracheids perform the 
double function of wood fibers and tracheal tubes. 

The Stone Cells. — The stone cells are formed by the thick- 
ening and lignification of the walls of originally thin- walled 
parenchyma cells ( Fig. 15). They may be found in the pri- 
mary and secondary cortex, pericycle, medullary rays and pith, 
in the integuments of many seeds, and in the shells and stones 
of nuts and stone fruits. In the latter instances they may 



THE STONE CELLS 



»5 



form continuous tissues, but in roots, stems and leaves they 
occur in more or less isolated groups or even singly. They 
occur in many barks in sufficient numbers to make them notably 
strong and hard, as in the hickory, and they sometimes reen- 
force the bast fiber tissues by forming firm unions between 
their separate strands, as in the oaks. They contribute to the 
tough and leathery character of some leaves, as in Camellia 
and Olea, for example, where they occur scattered amongst 
the mesophyll cells, to which they, indeed, belong morpholog- 
ically. Small groups of stone cells are found scattered through- 
out the pulp of such fruits as the pear and quince and they 
give to these their gritty character. 

The uses of stone cells are apparent. In nuts and stone 
fruits they box up, against injury and loss, the embryo and 
other tender parts of the seed, and are in such cases of the 
nature of an exoskeleton such as a turtle has among animals. 
In barks, fruits, and leaves, where they occur in more or less 




Fig. 38. Stone cells from different sources. 1, from coffee; 2, 3 and 4, from 
stem of clove; 5 and 6, from tea leaf; 7, 8 and 9, from powdered star- anise seed. 
(After Moeller.) 



86 THE PLANT SKELETON 

isolated groups they give hardness and toughness without 
being an impediment to increase in size. 

The stone cells are frequent and important landmarks in the 
study of powdered drugs and condiments. Fig. 38 shows a 
variety of forms, enough to give a general conception of their 
visible characters. 

Topography of the Skeleton. — Purely mechanical consid- 
erations can not alone be taken into account by plants in the 
location of the skeletal tissues. The trunk of a tree, for in- 
stance, is something more than a strong column to bear the 
crown aloft : it is also a part of the body through which water 
and food must circulate and be stored; respiration, digestion, 
and assimilation must occur in it as in other members of the 
plant ; and cell division and growth must take place there, and 
these functions are quite as important as that performed by 
the skeleton. Therefore the problem before plants in the 
building of their skeletons is to follow the best mechanical prin- 
ciples wherever this can be done without too great sacrifice of 
the other functions. 

In Dicotyledons it is of the utmost importance that the parts 
of the skeleton be so placed that they do not obstruct secondary 
increase in thickness ; while in Monocotyledons, where increase 
in thickness continues, as a rule, but for a brief period, this 
consideration is of much less importance. 

In stems, which have to bear the weight of the crown, and 
withstand the stretching and compressing stresses as they are 
swayed back and forth by the winds or other agents, the best 
position for the skeletal tissues from a purely mechanical stand- 
point is at the outside of all other tissues, in the form of a 
hollow cylinder ; or, other things interfering, as near this form 
and position as possible. But in all of the higher land plants 
it is an absolute necessity to have a tissue at the exterior suited 
to prevent loss of water, or to keep the water from filling the 
intercellular air spaces in case of the higher water plants, and 
so the skeleton must give way to the epidermis and cork. 
Further, in Dicotyledons, a complete skeletal cylinder at or 



TOPOGRAPHY OF SKELETON 87 

near the surface could not long- be maintained without its 
breaking asunder due to the new tissues formed by the cam- 
bium, or, if too strong for this, without preventing increase in 
diameter. These conditions, however, we find are happily 
met. Xext the epidermis is placed the collenchyma, in the 
form of a hollow cylinder or in separate strands. It is not too 
strong to resist growth in diameter ; and, since it is chiefly for 
temporary service until the bast and wood fibers have been laid 
down, it can without detriment be broken apart or tangentially 
stretched as growth in diameter proceeds. 

The bast fiber tissue is, as a rule near to the surface, but in 
isolated strands, in order that increase in diameter and the 
flow of water and sap radially to and fro may not be too much 
interfered with. The strands of bast fibers that stand in 
front of and against the phloem serve the double function of 
skeleton for the stem as a whole and for the thin-walled phloem 
tissues in particular which they sustain as the bones do the 
weaker tissues of the vertebrate body. In Fig. 17 different 
plans of collenchyma and bast fiber topography are shown. 

The secondary xylem in many herbaceous and all woody 
Dicotyledons and Gymnosperms furnishes a skeleton that is 
especially adapted to secondary increase in thickness, since it 
is located inside the cambium ring and can be increased indefi- 
nitely without hindering the growth of other tissues or being 
itself subjected to stresses that tend to tear it asunder. It can 
therefore form the continuous cylinder that is so highly desir- 
able in the plant skeleton. 

In many herbaceous plants the xylem cylinder surrounds a 
relatively large pith, and so conforms in the measure that other 
functions will permit to the accepted principles of mechanical 
construction where economy of materials is desired; and in 
woody perennials the xylem that is first formed is placed out- 
side a pith of greater or less dimensions, but secondary increase 
in thickness after a few years so far surpasses the original 
diameter of the stem that whether the xylem cylinder remains 
hollow or crowds in and crushes out the pith becomes a matter 
of no significance. 



88 



THE PLANT SKELETON 



?X c o^9- t ??gtg 




Fig. 39. Camera-lucida outline 
of portion of cross-section of 
corn-stalk, showing at g bast 
fiber zone beneath the epidermis 
and surrounding the outermost 
vascular bundles. 



In roots the xylem is placed closer to the center than in 
stems, and by crowding in very soon obliterates the pith and 

assumes the form of a solid rod. 
This difference of the wood skele- 
ton in the stem and the root is 
related to the difference in the 
direction of the stresses which 
they have to overcome. In the 
stem it is the weight of the crown 
and the alternate stretching and 
compressing when swaying in the 
wind; while in roots it is the pulling force which the swaying 
stem exerts on the roots, and the compressing force with which 
the soil resists growth in thickness of the roots, and both of 
these stresses the solid wood cylinder of roots is well adapted 
to withstand. 

In most monocotyledonous stems the problem of locating 
the skeletal tissues is simplified because no allowance needs to 
be made for secondary increase in the vascular bundles, and 
seldom for secondary increase in the stem as a whole, and this 
fact is taken advantage of by encasing, and so bracing and 
strengthening each vascular bundle in a sheath of scleren- 
chyma or bast fibers developed from the ground parenchyma 
tissues (Figs. 28 and 40). 

In grasses and similar Monocotyledons many vascular bun- 




g F «" d 

Fig. 40. Cross-section of a portion of palm stem, e, xylem; f, phloem portions of 
vascular bundle; g, sclerenchyma tissue about vascular bundle; d, fundamental or 
ground tissue; c, larger tracheal tubes in vascular bundle. (After Engler and 
Prantl.) 



TOPOGRAPH V OF SKELETON 



8 9 



dies are massed close to the epidermis, each with its protecting 
and strengthening sclerenchyma cylinder, and all are hound 
together by thick-walled ground parenchyma, and bundles of 
subepidermal bast fibers flank the most exterior bundles (Fig. 
39). These facts account for the hardness and strength of 

the exterior part of many grass 
stems, such as corn and bam- 
boo (Fig. 38). 

In palm stems the skeleton 
consists of numerous strands 
of bast fibres in the peripheral 
ground tissue, and large masses 
of these fibers of extraordi- 
nary hardness and strength 
surrounding each vascular 
bundle wherever located (Fig. 
40). There being no cam- 
bium, wood fibers are not pro- 
duced as in the Dicotyledons, 
and the xylem part of the vas- 
cular bundle is occupied by 
tracheal tubes, tracheids, and 
xylem parenchyma. 

In leaves the skeleton is in 
the form of bast fibers attend- 
ing the main ramifications and 
the vascular 
bundles ; and sometimes sub- 
epidermal bast strands occur at 
the edges of leaves or at other places without direct connection 
with the vascular bundles. Collenchyma is also sometimes 
employed to strengthen the leaf borders. 

Fig. 41 indicates diagrammatically the progress in the devel- 
opment of a woody plant. At and near the growing apex 
there are no skeletal tissues. Some distance back from the 




Fig. 41. Diagram showing the pro- 
gressive development of the skeletal tis- anaStOlTlOSeS of 
sues from the apex towards the base of 
the stem. 



90 THE PLANT SKELETON 

apex the collenchyma appears, and further back bast fibers and 
stone cells reenforce the collenchyma. Finally, in older parts 
still the wood fibers make their appearance and the wood zone 
is continually broadened by the activity of the cambium as the 
stem grows older. This stretches and breaks the collenchyma, 
and it, as well as the bast becomes gradually of less and less 
importance as the wood increases. 

Illustrative Studies 

i . We have already seen collenchyma in both cross and lon- 
gitudinal sections in the stem of Aristolochia ; we can, how- 
ever, find this tissue carried to a higher stage of development 
in the stem of sunflower, Zinnia, hemp, and many other her- 
baceous plants. Make cross-sections from such a plant and 
mount them in dilute glycerine. Note the great thickening of 
the walls at the angle of the cells. The shapes of the cells, 
as outlined by the primary walls, before thickening began, can 
be seen. Draw a few cells to scale. Measure the thickness of 
the walls across the corners. Are not these thickenings really 
vertical rods? Study longitudinal sections to see how con- 
tinuous these rods are. 

2. In the stem of Aristolochia we found a sclerenchyma 
ring made of long cells with walls lignified and somewhat 
thickened. These are bast-like, but they are not typical bast 
fibers. Sunflower and species of Abutilon, flax and hemp will 
furnish good examples of fibers of various lengths. 

Study cross-sections of sunflower stem in aniline sulphate 
(page 254). The bast fibers will be yellow. Or when the 
fibers are clearly recognized they may be studied in dilute 
glycerine. Draw a few cells to scale. Measure the thickness 
of their walls. Macerate longitudinal sections in sulphuric 
acid-alcohol and ammonia (see under Maceration in Chapter 
XV) and tease out the fibers in a drop of dilute glycerine. 
Draw a few fibers to scale and measure their lengths. 

Study cross-sections of young and old stem segments of 
Abutilon Avicennse mounted in aniline sulphate. Here the 



[LLUSTRATIVE STUDIES QI 

first-formed groups of bast fibers belong to the pericycle and 

later groups have descended from the cambium. Find the evi- 
dence for this. Draw a few cells from a mature group and 
others from a younger group where thickening of the cell walls 
is not yet complete. Macerate longitudinal strips of the bark 
In sulphuric acid-alcohol-ammonia, tease out some of the fibers 
in dilute glycerine, and draw one or more to scale. Measure 
the lengths of the fibers. 

Study cross-sections of flax in aniline sulphate. The sec- 
tions are best made from small lengths of stem imbedded in 
celloidine or collodion (page 233). In your judgment what 
is the origin of the numerous groups of fibers found here, 
From the ground meristem of the cortex? From the peri- 
cycle? From the cambium? The student should now be 
able to answer these questions from his own observations. 
Make a diagram showing the position and frequency of the 
groups. 

Macerate strips of bark as told above for Abutilon, only 
here the strips should be 5 or 6 cm. long. Tease out a single 
fiber and measure its length. Draw a portion of its length as 
seen under high magnification. 

3. Study cross-sections of seeds that have been soaked in 
water and imbedded in glycerine gum (see under this head in 
Chapter XV). Draw some of the cells to scale. Xote the 
character and frequency of pits in the walls. Treat some of 
the sections with phloroglucin (see under this head in Chapter 
XV). Are the walls of the stone cells lignified? 

Make thin sections of stone cell tissue from the stone of a 
peach and the shell of a cocoanut. This can be done by sawing 
as thin a section as possible with a hack saw, and then rubbing 
it to the requisite thinness between two water hones kept wet. 
This is a slow process and one is in danger of losing the section 
altogether towards the end of the operation; but sections ob- 
tained in this way are worth the labor. Another way to get 
sections that will do fairly well is to shave them off with a 
sharp knife. They will curl tightly when made in this way. 



92 THE PLANT SKELETON 

but the thinnest may be selected and forcibly straightened out 
in a drop of water. If they break in doing this, nevertheless 
the small fragments will show what is wanted. Stain the sec- 
tions in fuchsin or safranin, dehydrate them in alcohol, rinse 
them in xylene and mount them in balsam. 

4. Study cross-sections of pine wood in dilute glycerine. 
Note the absence of tracheal tubes and the presence of rela- 
tively large tracheids in the early growth and small ones in 
the late growth. Examine a longitudinal radial section and 
compare the tracheids of the early and late growths. Draw 
a few tracheids from the two regions from both points of view 
to show the relative sizes of the tracheids and wall thicknesses. 

Macerate longitudinal sections in nitric acid and potassium 
chlorate (page 279) and tease out the tracheids in dilute gly- 
cerine. Draw to scale a tracheid from the early and the late 
growths. 

Study in a similar manner wood of oak, walnut and yellow 
poplar. In both the macerated and unmacerated longitudinal 
sections note the difference between wood fibers and fiber 
tracheids, both similar in form, but the former with plain 
pits in the walls and the latter with bordered pits (page no). 

What does the microscopic examination show as the relative 
hardness and lightness and uniform grain of these woods? 

In the longitudinal sections and macerations medullary rays 
and wood parenchyma will be met with, and these should be 
studied here enough to recognize their characters, although a 
detailed study of them will be undertaken in another chapter. 



CHAPTER VI 

THE ABSORPTION OF WATER AND MINERALS 

All substances that penerate into the body of the plant cell 
must be in solution, excepting in the case of low forms of 
plants destitute of cell walls which are sometimes able to 
engulf solid particles. Simple unicellular and filamentous algae 
can absorb water throughout their entire surface, but more 
complex plants from the liverworts and mosses upwards to the 
seed plants, which have ventured to raise a part of their bodies 
above the substratum where the energy of the sunlight and 
materials of the atmosphere can be more freely appropriated, 
have found it necessary to put forth special absorbing organs 
into the substratum for the intake of water and minerals ; and 
the larger, taller, and more branched the part above the sub- 
stratum, the more extensive on the whole must be the absorb- 
ing parts beneath the substratum. 

From the vascular cryptogams (ferns, lycopods, equisetums) 
up through the seed plants, roots are employed for anchorage, 
and for absorption and conduction to the stem, of water and 
soil solutes. In floating water plants and in many aerophytes 
(air plants) there are roots that serve for absorption and con- 
duction only. 

Roots in the Soil. — It is only the younger parts of soil 
roots, and particularly the root hairs growing near their apices, 
that are fit to carry on absorption, the older parts having the 
walls of the exterior cells more or less waterproofed. As will 
presently be seen the root hairs are the solution of some diffi- 
cult problems in the relation of plants to the soil. In order to 
penetrate the soil in a necessarily sinuous course, and to get 
past obstructions in the best way the place of elongation in 
roots has been restricted to the region of the apex, so that this 

93 



94 



ABSORPTION OF WATER AND MINERALS 




Fig. 42. 



Cross-section of a root in the region 
of the root-hairs. 



delicate, sensitive part might feel its way amongst the soil 
particles as it elongated, without being shoved and jammed 
forward as would be the case if elongation were kept up in 
the remoter parts. 

Under average conditions the water in the soil exists as a 
thin film around each soil particle and holds in dilute solution 

(0.0 1 per cent, to 0.03 per 
cent.) some of the con- 
stituents of the particles 
that are necessary to 
plants. To get the water 
and solutes it is obviously 
necessary that many fine 
outgrowths from the root 
should reach out on all 
sides, and, pressing them- 
selves against the soil par- 
ticles, become immersed in the films of water. 

The Root Hairs. — The root elongates close to its apex, and 
2 or 3 mm. back from this it ceases to grow in length and some 
of the epidermal cells here grow out in the 
form of slender tubes known as root hairs 
(Fig. 42). So far as recorded measure- 
ments show these may become from a 
fraction of a millimeter to 8 millimeters in 
length. 

Growing only at its point a root hair 
reaches out through the humid atmosphere 
of the soil interspaces until it strikes a 
solid particle, when it bends about this 
and flattens out over it to a certain extent 
(Fig. 43). At the place of contact the 
delicate cellulose wall of the hair becomes somewhat mucilagi- 
nous, and is thus all the better able to cling on and imbibe 
water, and when it reaches a soil particle and becomes fastened 
to it this contact seems to act as a stimulus to stop its further 




Fig. 43. Apex of 
root-hair flattened out 
over and imbedding 
soil particles. 



THE ROOT HAIRS 



95 



growth in length. By means of the root hairs the roots are 
able not only to make close contact with the soil and soil water, 
but they also increase their absorbing surface many times — 
from five to twelve times according to recorded estimates. So 
that if the hairs were stripped away the capacity for absorp- 
tion would be correspondingly reduced. The effect of this we 
see when young plants are transplanted, for even when great 
care is taken not to break the roots wilting is apt to occur at 
first because the delicate root hairs have been torn off or have 

died away as a result of the shock of 

transplantation. Soon, however, new 
hairs are formed, water is again ab- 
sorbed in plenty, and the plant picks up 
from its wilted condition. 

The protoplast of the mature root 
hair is in the form of a very thin film 
lining the wall, and the cavity which 
it surrounds is filled with cell sap con- 
taining sugars, acids, etc., in solution 
that afford osmotic conditions for the 
intake of soil water with considerable 
power (Fig. 44). 

Any substances from the soil, either 
water or solutes, before mingling with 
the sap in the root hairs, must pass 
through the cellulose cell wall, the ex- 
ternal plasma membrane, the general 
cytoplasm, and the internal plasma 
membrane. The living plasmatic parts, 
however, taken altogether form a film 
so thin as to be discerned with difficulty 
even with high powers of the micro- 
scope. Not all substances in solution in the soil water are 
able to make this passage. Probably all of them can pene- 
trate the cellulose wall, but to some the external plasma mem- 
brane presents an impassable barrier, and this membrane is 




Fig. 44. A single root- 
hair on a large scale, 
showing that it is an out- 
growth of an epidermal 
cell, and the fact that it 
possesses a living proto- 
plast and large vacuole 
filled with cell sap and 
traversed by cytoplasmic 
strands. The nucleus is 
near the apex of the hair. 



g6 ABSORPTION OF WATER AND MINERALS 

then said to exercise a selective function. Similarly the plasma 
membranes, external and internal, keep the osmotic and nutrient 
substances of the cell sap from escaping. We must not, how- 
ever, overlook the fact that substances that have penetrated 
the wall of the root hair but are unable to pass the plasma 
membrane may by travelling in the wall alone, enter the plant 
body and become distributed throughout its length without 
once having entered the living cells. 

The selective action of the plasma membranes has never 
been satisfactorily accounted for on the basis of chemical and 
physical processes alone, although, presumably, this could 
be done if all the conditions were accurately understood. 
As soon as the protoplast dies the membranes lose their 
power of selection and the cell sap readily escapes from 
the cell; and so it seems the membranes are able to 
do their work because they are living. When we are unable 
to give a chemical or physical explanation of a physiological 
phenomenon of this sort we speak of it as due to a vital power, 
by which we mean that its seat is in the living protoplasm and 
its origin is shrouded in mystery. And the mystery in this 
instance is the more profound because the selective action 
varies in purposeful ways with the self -regulatory action of 
the protoplast, as will be brought out in subsequent chapters. 

The root hairs excrete organic acids and carbon-dioxide, and 
these go into solution in the soil water and have a solvent effect 
on some of the soil constituents. 

The water and solutes (substances in solution in the water) 
absorbed by the root hairs pass into the adjoining cortex cells 
and thence across the root to the tracheal tubes and tracheids 
of the xylem where they begin their ascent into the stem. The 
relative positions of the primary xylem and phloem strands 
(Fig. 42) which make it possible for the water and solutes 
(now after their entrance into the plant called the crude sap) 
to reach the xylem without traversing the phloem is evidently 
a device to keep the crude sap and the elaborated sap (sap con- 
taining soluble food such as sugar in solution) distinct and 



METHOD OF INTAKE OF WATER 97 

apart. As will be seen in Chapter X the elaborated sap makes 
its way in the phloem longitudinally throughout the plant from 
the leaves to the roots. 

The root hairs are very short lived and the old ones die 
away about as fast as the new appear. After the root hairs 
die the walls of the outer cortex cells become more or less 
suberized and by this are made stronger and better protected 
against parasites, but less able to take in water and solutes. 
Probably all of the cells of the root epidermis are, up to a cer- 
tain age, capable of growing forth as root hairs, and only need 
the stimulus to do so. for we find that the number of root hairs 
varies with the character of the environment, more being 
formed where more are needed. For instance, when grown 
in a moist atmosphere, where only that water can be absorbed 
which the air holds, the hairs are very numerous and long, 
while in a moist soil where the hairs can become partly sub- 
merged in the water films about the soil particles shorter hairs 
in less number are formed ; and where roots are grown in water 
fewer and shorter hairs grow T forth or none at all. 

Method of Intake of Water and Solutes. — The water and 
solutes enter the root hairs by osmosis and diffusion. The 
sap of the root hairs holds in solution osmotic substances such 
as sugar and acids which cause the inflow of the water, and 
since this is continually passed on to the conducting tissues the 
conditions causing its intake are more or less constant. The 
substances in solution in the soil water (the solutes) pass into 
the root hair by diffusion, and the speed of their onward move- 
ment through the membranes is by no means necessarily the 
same as the rate of inflow of the w T ater. If the intake of water 
is accelerated because increased evaporation from the leaves is 
creating larger demands for water, it does not follow that the 
entrance of solutes into the root hairs is hastened to the same 
extent. The movements of water and solutes are governed by 
different conditions. The solutes keep going in so long as 
there is less of them in a unit of volume inside the cell than 
outside; while the water continues to enter while there is a 



9$ ABSORPTION OF WATER AND MINERALS 

greater concentration of osmotic substances inside than out- 
side. And the same thing is true in the interchange of water 
and solutes between different cells of the plant body, namely 
the water passes from the cell having less into the one having 
the greater concentration of solutes, while the solutes pass from 
the cell having greater into the one having the less concentra- 
tion of solutes. Of course, however, inside a cell or water 
tube the water tends to sweep the solutes along in its currents. 

It appears that so long as the osmotic conditions are good 
the water can pass freely into the plant and from cell to 
cell ; but this is not true of all solutes, for the plasmatic mem- 
branes will not let all kinds pass, although many kinds are 
allowed to enter that are apparently of no use. As has been 
said it is not understood just how the membranes act in 
discriminating between solutes ; but the size of the molecules 
of the latter apparently is not always a decisive factor, for 
large molecules are known to enter when smaller ones are held 
back. The plasmatic membranes are however subject to 
change and may at one time allow a certain substance to pass 
and not at another. 

Effect of Temperature of Soil, and Character and 
Amount of Solutes upon Absorption. — A warm soil is con- 
ducive to rapid absorption, while a cold soil hinders and may 
practically stop it ; although a perceptible amount of absorption 
may in some cases still take place at or below o° C. The cold- 
ness of the soils of arctic and alpine regions acting in this way 
has a very great influence on the stunted growth and econom- 
ical use of water of the plants native to these places. 

If the soluble salts in the soil reach a concentration above 
.5 per cent, they hinder the flow of water into the plant either 
by their influence on osmosis or by a poisonous effect on the 
absorbing organs. Thus it comes about that plants along the 
strand or in salt marshes have reduced transpiring surfaces 
correlated with the slow intake of water, as seen in the Russian 
thistle and garden asparagus, both of which are native to the 
salty soil of seacoasts. Humic acid also, which is formed by 



ABSORPTION OF WATER BY AEROPHYTES 



99 



the decay of vegetation in boggy places, retards the absorption 
of water enough to call forth a reduction of the transpiring 
surfaces. 

Absorption of Water and Solutes by Aerophytes. — The 
aerophytes, epiphytes, or air plants, send no roots into the soil, 
nor parasitic roots into the plants on which they have found 
lodgment; but they derive 
all of their supplies from the 
atmosphere, which contrib- 
utes its moisture in the form 
of rain or dew, provides 
carbon dioxide and oxygen 
just as it does for other 
green plants, and brings soil 
substance in the form of dust. 
These plants are not there- 
fore living on the air alone, 
as their name might imply, 
but they have essentially the 
same raw materials for 
building their food as have 
plants rooted in the soil. 

To illustrate the essential 
mode of their obtaining wa- 
ter and solutes two types of 
aerophytes will be discussed, 
namely, the type where true 
roots are sent forth into the 
air, and the type which, 
producing no roots, has its 
stems and leaves equipped 
for absorption. In the first instance, some tropical orchids, 
and other aerophytes lodging on the branches of trees, send 
forth roots into the air that have an external covering of dead 
tissue known as the velamen (Fig. 45). The cells of this tis- 




Fig. 45. Portion of a cross-section 
through an aerial root of Stanhopea ocu- 
lata. h, the velamen; i, exoderrais; ;', 
cortex; A', endodermis. (After Haber- 
landt.) 



IOQ 



ABSORPTION OF WATER AND MINERALS 



sue have many very minute openings through their exterior 
and interior walls through which water passes when the root 
is wet with rain or dew. The velamen is formed by tangential 
division of the protoderm, beginning a short distance from the 
apex and giving rise to layers of cells varying from one to 
eighteen or more according to the species. The cell walls of 
the velamen sometimes remain thin, but usually they are thick- 
ened, either uniformly, or in the form of a network or spiral 
bands. After the cells have reached maturity the protoplasts 
soon die, and the cell cavities become alternately filled with air 
and water as a dry interval is succeeded by a wet one. The 
necessary soil constituents are doubtless obtained from the 
dust which gathers on the roots or is washed down to them 
from the other parts or from overhanging branches of the tree 
on which the aerophyte is encamped ; and from particles washed 
out of the atmosphere by the falling rain drops. 

Separating the velamen from 
the rest of the root is a cell 
layer known as the exodermis 
(Fig. 45) which is similar to 
the endodermis (page 35) of 
ordinary roots in having the 
cell walls more or less thickened 
and suberized, with the excep- 
tion of cells at intervals whose 
thin cellulose walls permit the 
passage of water and solutes 
which the velamen has gathered 
in. In some instances the outer wall of these passage cells is cov- 
ered externally by a felty mass of interlaced fibrous outgrowths 
(Fig. 46). It has been conjectured that this is a device to 
condense moisture from the atmosphere when rain and dew are 
not keeping the velamen supplied; but conclusive evidence is 
lacking to show that the velamen has the power to condense 
water from the vapor state by this or any other device. In 
any event the felty covering may help to retard evaporation 




Fig. 46. Showing at f the felty 
body covering the passage way through 
the exodermis of the aerial root of 
Sobralia macrantha. (After Haber- 
landt.) 



ABSORPTION OF WATER BY AEROPHYTES IOI 

through the walls of the passage cells when the velamen is dry. 
When the velamen is wet the root is as it embedded in a sat- 
urated sponge, and when dry the velamen acts as a mulch to 
keep the rest of the root from drying. 

Tillandsia usneoides, the hanging moss of the southern 
states, represents the second class of aerophytes where the 
roots do not develop, although their fundaments are present 




Fig. 47. Cross-section through a water-absorbing scale of Tillandsia usneoides; 
a, a, water- absorbing cells partially filled with water. (After Schimper.) 



in the young seedlings. This plant hangs from trees of vari- 
ous kinds, but has no organic connection with them and derives 
no materials from them. The branches are wiry and the 
leaves slender, and both are thickly beset with overlapping 
scales under which rain and dew gather and find entrance by 
osmosis into the cell cavities. Here the scales, like the vela- 
men, serve both for the absorption of water and protection 
against its loss. The scales when dry are shrunken and lie 
close against the stem or leaf ; but when wet their thicker outer 
wall swells and bulges outward, water is drawn into the cell 
cavities, and by their turgidity the scales rise and make room 
for more water beneath them (Fig. 47). Practically the 
whole plant body is thus enabled to imbibe water, and solutes 
that have come in the form of dust. 

Other Methods of Absorbing Water. — Some desert 
plants have devices for absorbing water into the leaves. 
Diplotaxis Harra, for example, a cruciferous plant of the 
Egyptian and Arabian deserts, has its foliage beset with 
stiff hairs which, acting as points for the radiation of heat, 



102 



ABSORPTION OF WATER AND MINERALS 




after sunset gather dew. The hair is practically waterproof 
excepting at its base, where the dew, running down from 
above, forms a film over the wall and is quickly absorbed. 

There are some interesting anatomical details in these hairs 
of Diplotaxis (Fig. 48). Cellulose additions to the wall fill 
the cell cavity down to the spreading base, where the cavity 
enlarges and is lined with an unusually 
thick protoplastic layer. The wall sepa- 
rating the hair from the body of the leaf 
has many pits through which the imbibed 
water can pass into a water reservoir tis- 
sue beneath, whence it is distributed di- 
rectly to the mesophyll cells. The entire 
leaf is so thoroughly waterproofed that 
only the basal part of the hairs can be 
wetted. Nevertheless when a wilted leaf 
is submerged in water it soon regains its 
turgidity. 

Practically the same device with modi- 
fied details is repeated in various other 
desert plants whose roots do not go deep 
enough to draw water from the depths of 
the soil. 
Although there are many devices for absorbing water and 
solutes under various environments, in one respect they are all 
alike : they are outgrowths that increase the absorbing surface 
many-fold and enable the plant to make effective demands on 
the source of supply. 



Fig. 48. Water- 
absorbing hair of 
Diplotaxis Harra. a, 
secondary cellulose 
thickening of the cell 
wall, filling the cell 
cavity nearly to base 
of hair; b, water- 
storage cells commu- 
nicating by means of 
pits with the cell- 
lumen of the hair. 
(After Haberlandt.) 



Illustrative Studies 
Soak a flower pot in water. Soak mustard seeds in water 
over night. Dash these seeds against the inner surface of the 
moist pot where they will stick because of their mucilaginous 
surface. Invert the pot in a saucer of water and the seeds will 
germinate and furnish an abundance of root hairs. Cut off 
the roots and lay them in a dish of 5 per cent. KOH over night 



ILLUSTRATIVE STUDIES I 03 

to bleach and clear them up. Mount one of the cleared roots in 
a drop of the KOH solution under a cover-glass and crush the 
root a little by gentle pressure on the cover-glass. Study with 
low and high powers. Can you now make out that a root 
hair is an outgrowth of an epidermal cell ? Measure the length 
and breadth of a hair and the thickness of its walls. Note the 
tracheal tubes near the center of the root. Draw an entire 
root hair including the enlarged basal part. Study a cross- 
section of a root put up in the form of a double-stained perma- 
nent mount as described in Chapter XIV. Find the stumps of 
root hairs. Note the number and character of the cells which 
the water and solutes absorbed by the root hairs must traverse 
in going to the tracheal tubes. Draw a segment of the cross- 
section to show all this. 

3. Make permanent double-stained mounts from paraffin 
material of Cuscuta parasitic on balsam or on any other host 
suitable to paraffin sectioning. Study with low and high 
powers. Note whether the water and food-conducting tissues 
of the parasite are in organic union with the corresponding 
tissues of the host. Can you tell precisely where the tissues 
of the parasite leave off and those of the host begin? and if so, 
how can it be told? Show by a diagrammatic drawing the 
host and parasite, and on a larger scale draw a few cells of the 
phloem and xylem of the host and corresponding cells of the 
parasite joining these. 

4. Study free-hand cross-sections, or paraffin sections done 
into permanent mounts, of Tillandsia usneoides. Mount free- 
hand sections in dilute glycerine. Make a drawing through a 
scale showing its relation to the tissues of the stem. 



CHAPTER VII 

CIRCULATION OF WATER AND SOIL SOLUTES 

The Need of a Circulatory System. — Unicellular plants 
living in water or in moist and shady places can absorb water 
and solutes throughout their entire surface, and in simple mul- 
ticellular water plants, consisting of a single row of cells or 
of an expanse of tissue only one cell in thickness, each cell is 
in position to absorb water and solutes for itself. But in 
bulkier plants the interior cells have to draw upon the exterior 
for their necessary materials. And where the distance to the 
remoter cells is great, and loss of water through leaves and 
other above-ground parts is considerable a system for the con- 
duction of water and solutes becomes imperative. 

A tissue made up of short cells will not serve this conductive 
purpose, as is shown by the fact that a strip of pith or cortex 
with its lower end in water will soon begin to wither at a 
height of five to fifteen centimeters, even when protected from 
rapid loss of water ; and experiments involving the extirpation 
of the pith and the removal of the bark show that the water 
tubes in a few thin vascular bundles can supply the water lost 
by transpiration through the leaves, while all of the tissues of 
the pith and bark combined fail to do this. These things show 
us how necessary has been the differentiation of a water-con- 
ducting system as plants in their evolution have aspired to 
greater and greater heights above the soil. 

Tissues Devoted to the Conduction of Water. — The 
tracheal tubes and tracheids have been shown to be the high- 
ways through which water and solutes that have entered from 
the soil make their way to the leaves. The origin and nature 
of these tissues have already been told in Chapters II and III, 
and we have now to consider their structural adaptations to 
the work they have to do. 

104 



THE TRACHEAL TUBES 105 

The Tracheal Tubes. — The tracheal tubes, or water tubes, 
are fitted for carrying- water by being essentially continuous 
tubes from the finest branches of the roots up through the 
stem and into and throughout the leaves. In some instances 
cross walls have been found in them every 45 to 91 centimeters 
apart, but the amount of resistance which these walls afford to 
the ascent of water must be extremely slight compared to that 
which would come from the 15,000 to 30,000 cross walls in 
a chain of ordinary cells reaching to the same height. 

The average diameter of the tracheal tubes is approximately 
.05 mm. They average the smallest in submerged water 
plants where the need for them is not great, and the largest 
in tall-growing climbing or clambering plants where stems of 
small diameters have to carry relatively large amounts of water 
through long distances. In shrubs and trees the water tubes 
are larger in the early growth of spring and summer, when 
the new crop of leaves is to be supplied with water, than in 
the later growth when the demand for a larger water-carrying 
capacity has been almost or quite satisfied. 

It has been told in Chapter II that the walls of the tra- 
cheal tubes are thickened after various patterns, spirally and 
annularly (Fig. 19) in those that are first formed by the pro- 
cambium, and more extensively, leaving thin places in the form 
of circular or transversely elongated pits in those that are last 
formed by the procambium and later by the cambium (Fig. 
19). The thickenings of the walls are needed to hold the 
tubes open against growth and turgor pressures from the sur- 
rounding tissues, pressures great enough to burst the bark even 
when this is reenforced by a continuous sclerenchyma ring, 
as in the case of Aristolochia (compare Figs. 23 and 24), and 
to crowd the wood in to the complete obliteration of the pith. 

The thin places between the spiral and annular thickenings 
and in the pits are of course needful to allow water and solutes 
readily to pass out to supply the surrounding tissues as they 
need them, and to permit reserve food solutes to pass in when 
after a period of storage, in the wood parenchyma and medul- 



106 CIRCULATION OF WATER AND SOIL SOLUTES 

lary rays, they are needed above by unfolding buds and devel- 
oping seeds and fruits. 

The spiral and annular thickenings are such a nice adjust- 
ment to specific conditions that their significance, already men- 
tioned in Chapter II, will admit of further discussion. Tubes 
of these kinds are the first permanent tissues to become differ- 
entiated from the procambium, and they will be found in stem 
cross-sections not far from the growing apex and on the side 
of the procambium bordering the pith. Already before other 
tissues of the vascular bundle make their appearance in the 
procambium strand these tubes have characteristically thick- 
ened and lignified their walls. In longitudinal sections they 
stand out sharply as channels for conduction of water into the 
meristematic region close to the growing apex. Where these 
tubes appear a good deal of stem elongation has yet to take 
place, and it will be seen at once that wall thickenings in the 
form of rings or spiral bands will allow the tubes to stretch 
without interposing much resistance to stem elongation. The 
pitted kinds of tubes which later appear are formed farther 
back from the apex where growth in length has ceased, and 
these are stronger and better adapted for long service than are 
the earlier sorts which finally become ruptured and ineffective. 

Course of Tracheal Tubes Through the Stem.— The 
course of the tracheal tubes through the stem is best followed 
by tracing the course of the vascular bundles of which they 
form a part. Nearly all vascular bundles end in the leaves, 
and only rarely do they end in the stem without entering a 
leaf. Therefore there is no better way to trace the bundles 
than to begin in the leaves and follow their course downwards. 
Proceeding in this manner we find that the bundles or bundle, 
as the case may be, that descend into the stem from each leaf 
continue their downward course through one or more inter- 
nodes, and then as a rule branch and fuse with bundles that 
have entered) the stem from other and lower leaves (see Figs. 
49 and 50). In this way the whole system of bundles in 
stems is made continuous, a system comparable in this respect 
to the anastomosing veins and arteries of the animal body. 



COURSE OF TRACHEAL TUBES THROUGH STEM 



07 



It will be seen that if the leaves on one side of a tree are 
made to transpire faster than those on the other because of 
greater exposure to the sun or drying wind from that quarter 
they can draw upon the water in the whole circle of bundles 
in the trunk; or when one side of a stem is severely injured 
so as to interrupt the flow of water on that side, the leaves 
above the other part can draw their water from the opposite 
side, which in turn can 
reimburse itself from 
the injured side below 
the wound. 

The vascular bundles 
that descend from a 
leaf into the stem con- 
stitute a leaf trace. The 
number of bundles in 2T 
each leaf trace varies 
commonly from one to 
three, but there may be 
more; The rule is that 
the whole ring of vas- 
cular bundles in Di- 
cotyledons is composed 
of leaf traces, but, as 
was stated above, in- 
stances occur where 
bundles do not extend 

Fig. 49. A, diagram of course of vascular bun- 

mtO the leaves, and these dies in a palm stem; im, 2m, 3m, bundles from the 

11 ,1 median portions of the leaves. B, diagram of vas- 

anOmaiOUSly are in tne cular bundles in external view and in cross-section 

pith Or even in the COr- °^ t ^ ie stem °* Cerastium. The leaves are shown 

cut off at 1, 2, 2' and 3. (After Vines.) 

tex, and since they do 

not run out of the stem they are called cauline bundles, while 
the others that run into the leaves are called common bundles. 
The vascular bundles in most monocotyledonous stems are 
all leaf traces. It will be remembered that the leaves of these 
plants are parallel-veined, and usually many bundles descend 




io8 



CIRCULATION OF WATER AND SOIL SOLUTES 



from a single leaf into the stem. 




Fig. 50. Diagram showing the course of 
the vascular bundles in a stem of Clematis 
viticella. Median bundles from the leaves are 
marked A, lateral bundles B and C. (After 
Nageli.) 



These at first penetrate 
rather sharply towards the 
center, and then descend- 
ing turn gradually out- 
wards and unite with 
bundles from other leaves. 
The larger bundles go 
farther towards the center 
than the weaker ; and so it 
happens that the bundles 
in most Monocotyledons 
are not arranged in a con- 
centric circle as in Dicoty- 
ledons, but are more or 
less promiscuously dis- 
tributed, as seen in the 
stem cross-section ( Fig. 
49). Monocotyledons 
with hollow stems vary 
from this plan in that the 
bundles keep closer to the 
periphery ; and in the grass 
type of stem the bundles 
are united at the nodes by 
a plexus of anastomosing 
branches. 

Throughout all classes 
of stems it is a noteworthy 
fact that the leaf has one 
or more vascular bundles 
descending into the stem 
to take on and conduct to 
it directly the water and 
solutes which it as a trans- 
piring and photosynthe- 
sizing member demands. 



THE TRACHEIDS I O9 

The Tracheids. — The tracheids are elongated cells espe- 
cially adapted to be water carriers by numerous thin places 
in the walls in the form of bordered pits or associated with 
spiral, annular, or reticulate thickenings (Fig. 19). Each 
tracheid is a single closed cell, and water and solutes flowing 
into and out of it find the thin places suited to their passage. 

The length of the tracheids varies a good deal ; from one to 
two millimeters is a common length, but they are often shorter, 
and they may become many times longer than this. It there- 
fore appears that a water-conducting system composed of tra- 
cheids would offer more resistance to the flow of water than 
one made up chiefly of tracheal tubes, because of the less fre- 
quent cross walls in the latter case. 

Tracheids commonly occur associated with the tracheal tubes, 
but they are sometimes lacking. They usually replace the 
tracheal tubes in the smaller ramifications of the veins of leaves, 
and where tracheal tubes anastomose, as notably at the nodes 
of grasses, the connections are commonly made by tracheids ; 
and frequently tracheal tubes in successive annual rings of 
growth communicate by means of tracheids. 

Comparing the use of tracheal tubes and tracheids among 
the Monocotyledons and Dicotyledons it appears that the tra- 
cheal tubes are most in use for conduction through long dis- 
tances, while for transport through short distances the tra- 
cheids are preferred. 

In Pteridophytes tracheids occur more frequently than tra- 
cheal tubes, and in Conifers tracheids constitute the sole water- 
conducting tissue, excepting special water-conducting paren- 
chyma in the medullary rays; and the nearest approach to 
tracheal tubes is found in the elongated, spirally-thickened 
tracheids, which as a product of the procambium alone, occur 
next the pith. 

The wood of the pine, save the medullary rays, and a small 
amount of w r ood parenchyma devoted to the secretion of resin, 
is composed entirely of fiber-tracheids which serve perfectly 
well for the conduction of water, even to the tops of tall trees. 



no 



CIRCULATION OF WATER AND SOIL SOLUTES 



It does not necessarily follow, however, that tracheids are as 
efficient in the transport of water as are tracheal tubes, for 
under like conditions the leaves of pine transpire very little 
water compared with the leaves of ordinary dicotyledonous 
trees. The leaves of the beech, for example, weight for 
weight, give off ten times as much water as do those of the 
pine. The pine tree relies upon these fiber-tracheids for 
strength as well as for the conduction of water, and the success 
of the plan is shown by the fact that pines grow to be great 
trees and thrive in exposed situations where the stress of the 
winds and demands for water are heavy. The essential details 
leading to the success of this plan appear to be these: First, 
as to water-conduction, practically the whole of the early 
growth is devoted to this, and the late growth materially 
assists, and the xerophytic leaves keep transpiration down to 
a minimum. Second, as to strength, the thin places in the 
tracheids are reenforced by prominently overhanging borders, 
and the two weakest tissues in wood, namely the wood paren- 
chyma and the tracheal tubes are, respectively, almost and en- 




^ar — 




m 1 'ii'i'ii 

i 

Mil wily 7 


1 Hi,hM%, 


w 


1 

m 



Fig. si. Different stages in the development of a bordered pit. b, the original, 
thin, primary wall; a, the overhanging border formed as the wall thickened. B, 
thickening of the wall has continued and extended the border; the primary wall 
has thickened at c, forming the torus. C, the border and the torus are finished. 



tirely lacking. Considered as strengthening elements the fiber- 
tracheids are in all essential respects like wood fibers. They 
are long and tapering cells, their ends interlace, their walls are 
thickened and lignified. But in one noteworthy detail they 
are very unlike wood fibers, namely, their walls have numerous 



THE TRACHEIDS 



I I I 



and large bordered pits, and it is this that fits them for water 
highways (Fig. 51). 

The bordered pit is a thin area in the wall with an over- 
hanging border. The thin place is clearly to facilitate the 
passage of water and solutes, and the border serves the double 
purpose of strengthening the wall where it is weakened by the 




Fig. 52. Diagrammatic representation of a block of pine wood highly magnified. 
a, early growth; b, late growth; c, intercellular space; d, bordered pit in tangential 
wall of late growth; m, f and e, bordered pit in radial wall of early growth from 
different points of view; h, row of medullary cells for carrying food; g, row of 
medullary ray cells for carrying water; k, thin place in radial wall of ray cells that 
carry food. 



112 



CIRCULATION OF WATER AND SOIL SOLUTES 




Fig. 53. Diagram to show 
the tangential flow of water 
from the side where there is 



thin place, and of arresting and bracing the thin part when 
from unequal pressure at one side or the other it is in danger 
of bulging too far and bursting. 

Wlndward In the early growth of the annual 

ring of pine the bordered pits are 
almost exclusively in the radial walls, 
while in the late growth they occur 
for the most part in the tangential 
walls (Fig. 52). This difference 
seems to relate to certain physio- 
logical demands. If for any reason 
one side of the tree — the windward 
side, for instance — demands more 
water than the others, the water ris- 

less demand to the side where ing through the trunk Can paSS tO 
the demand is greater. , . 1 , 1 . 

that side through the pits in the radial 
walls, as shown in the diagram (Fig. 53). And since the med- 
ullary rays extend individually but a fraction of a millimeter 
vertically the water can find its way around the stem without 
traversing them, as illustrated by 
Fig. 54. Again, when the new 
crop of leaves is produced in the 
spring and the cambium commen- 
ces the new ring of growth, begin- 
ning in the crown and progressing 
towards the roots, the bordered 
pits in the tangential walls of the 
late growth of the previous sum- 
mer permit water to pass into the 
tracheids of the new growth as 
the latter progresses towards the 

the roots and is not yet in position to draw directly upon the 
roots themselves (Fig. 55). 

How the bordered pits assist in the flow of the water ver- 
tically, which without doubt is their chief function, is made 
clear by a study of longitudinal tangential sections where it 




Fig. 54. Diagram showing by 
the arrows how the water can 
flow tangentially around a plant 
without traversing the medullary 
rays, which are indicated in black. 



TRACHEAL TISSUES AND MEDULLARY RAYS 



Tangential Wall 




Fig. 55. Diagram to show 
the radial flow of water in 
pine wood, from the tra- 
cheids of the late growth of 
one year into those of the 
early growth of the succeed- 
ing year. 



is seen that the pits are plentiful in the slanting common wall 
between two vertically contiguous 
tracheids (Fig. 56). 

Relation of the Tracheal Tissues 
to the Medullary Rays and Wood 
Parenchyma. — One of the functions 
of the medullary rays is to carry ra- 
dially into the bark water which they 
ha\'e taken on from the tracheal tubes 
and tracheids. Their other most 
Important function of transporting 
and storing food will be spoken of 
in later chapters. Wherever a medullary ray comes into 
contact with a tracheal tube or tracheid there are pits or 
thin areas in the common wall separating 
them through which water and solutes can 
the more easily pass. And the same con- 
dition exists where the wood parenchyma 
cells come into contact with the tracheal 
tubes and tracheids. The medullary rays 
and wood parenchyma are therefore in posi- 
tion to take up and store water, and to 
assist in its radial and tangential distri- 
bution. 

It has not been demonstrated, and it is 
indeed doubtful, that the rays and wood 
parenchyma as living tissues assist mate- 
rially the dead tracheal tissues in the ver- 
tical transmission of water. The undoubted 
use of their intimate relationship, aside from 
the radial and tangential distribution of 
water, will be told in subsequent chapters. 

The Ring of Annual Growth. — The 
physiological significance of the ring of 
growth has already been told in the chap- 
ter on secondary increase in thickness. Recapitulating in a 
9 




Fig. 56. Diagram 
indicating by arrows 
how the water in the 
tracheids of pine 
passes longitudinally 
from one tracheid to 
another. 



114 CIRCULATION OF WATER AND SOIL SOLUTES 

sentence : The large tracheal tubes and tracheids of the early 
growth provide for the increased demand for water, while the 
predominating wood fibers and smaller tracheids of the later 
growth are a response to the demand for greater strength. 
The ring of growth in its relation to the transport of water and 
solutes demands our attention in this chapter. 

The large tracheal tubes and tracheids of the early growth 
are the most efficient in carrying the transpiration stream 
because of their relatively large diameters. That, however, 
the tracheal tissues of the late growth are useful in carrying 
water is shown by experiments* with colored solutions, and by 
the numerous pits in their tangential walls which are evidently 
designed to deliver water to the early tracheal elements of the 
succeeding year. Furthermore there is frequent communica- 
tion of the late tracheal elements with the early elements of the 
following year by means of radial rows of tracheids; and at 
the close of the season's growth groups of small tracheal tubes 
are often produced which are to lie immediately against, or in 
close juxtaposition to, the large tracheal tubes of the succeed- 
ing year. 

The number of years the tracheal elements remain active 
varies with different species. In trees like the oak, walnut, 
etc., whose wood is differentiated into sapwood and heartwood 
the heartwood has lost the power to conduct water through the 
filling of the tracheal tubes by ingrowths of the wood paren- 
chyma into their cavities, and by the infiltration of the walls 
of the tissues in general with gums, resins, and coloring mat- 
ters. On the other hand, in trees like the beech which produce 
no heartwood the water highways remain functional through 
many years. But always it is the wood of the current year 
that is the most active in water conduction, while the part 
played by the older rings of growth lessens with each succeed- 
ing year. In perennial monocotyledonous stems, as in the case 
of the palm, for example, where there is no annual increase in 
the size of the bundles, the same tracheal elements must retain 
thir activity indefinitely. 



RINGS OF GROWTH AND GROWTH IN LENGTH 



15 



Relation of Rings of Growth to Growth in Length.— 
When the terminal bud unfolds and adds another year's growth 
in length to the stem, it, together with the new shoots from 
lateral buds, produces the leaves of the 
current year. All of the leaves, it will 
be remembered, with the exception of 
perennial leaves such as those of the 
pine, are borne on these new shoots. 
We must now proceed to enquire what 
is the relation of the tracheal elements 
of the new segments of stem to those 
of the leaves and of the preceding years' 
growths ? 

While the internodes of the new 
shoot are still elongating and the cam- 
bium has not yet begun secondary in- 
crease in thickness, only the small and 
few spiral and annular tracheal tubes 
differentiated from the procambium are 
present in the new growth to take up and 
carry water from last year's segment of 
stem into the unfolding leaves of the new 
shoot (Fig. 57). And it sometimes 
happens that the leaves are more than 
half grown before these first tracheal 
tubes are reenforced by others from the 
cambium. In this fact we have the evi- 
dence, since young leaves are known to 
transpire water rapidly, that the spiral 
and annular tracheal elements first laid 
down are very efficient water conduc- 
tors, through short distances at least. 
Soon, however, these earliest tracheal 
elements are reenforced by others laid 
down by the cambium, and on account 




Fig. 57. Diagram show- 
ing the relation of the 
water-carrying tissues of 
the leaves to those of the 
stem, and how the older 
rings of growth give up 
their water to the newer 
before this water can enter 
the leaves. The figures at 
the bottom indicate in 
years the age of the rings. 






Il6 CIRCULATION OF WATER AND SOIL SOLUTES 

of their weakness they break apart and go out of function 
(Fig. 57). 

The tracheal elements of the new shoot (new stem and 
leaves) have direct communication with the roots through the 
tracheal elements of the new ring of growth which the cam- 
bium adds along the whole stem. It often happens, however, 
that before the cambium has made this connection the leaves 
are already well along in their development and are drawing 
water through the channels of the older wood. How this is 
accomplished will be seen in Fig. 57. In this figure the spiral 
and annular tracheal elements are represented by a spiral line, 
the current ring of growth is the outermost zone with the 
ascending arrows and the older annular rings are between this 
and the pith. It will be seen that the first- formed tracheal ele- 
ments (the spiral lines in the figure) from the leaf join with 
those of the current year's segment of stem. It will be seen 
also that the first- formed tracheal elements in last year's seg- 
ment of stem, as well as of preceding years, have broken apart 
and presumably can be no longer functional. Before the cam- 
bium becomes active in the new shoot practically all of the 
water which the leaves get must be drawn through the small 
spiral and annular tubes of the new shoot from the tracheal 
elements of last year's growth. When the cambium begins 
its activity it forms new tracheal elements in the new shoot 
which unite with the tracheal tissues from the leaf and pursue 
a continuous course through the new ring of growth all the 
way down the stem and into the roots (Fig. 57). 

Relation of Annual Rings to the Leaves. — A study of the 
diagram, Fig. 57, will show how the tracheal elements from 
the leaves have most extensive and direct connection with the 
tracheal elements formed by the cambium the current year, 
and therefore the great advantage to water-conduction which 
comes from the formation, first of all, of large tracheal ele- 
ments in this new growth. 

Experiments with eosin and other aniline dye solutions have 
shown that water rises throughout the sapwood, but most rap- 



DISTRIBUTION OF WATER THROUGHOUT LEAF 



117 



idly in the newest rings, and that if the latter are cut through 

the older rings are then employed to carry the water past the 
gap, above which the water is distributed to the newer rings 
again for farther transportation. See basal part of Fig. 57. 




Fig. 58. Camera-lucida drawing of a bleached leaf of a Dicotyledon, showing the 
course of the vascular bundles, and how they end free in the mesophyll. B, the 
same for a leaf of a Monocotyledon, showing the anastomosis of the parallel veins by 
means of slender lateral branches; C, magnified detail of A; D, magnified detail of 3. 



Distribution of Water and Solutes throughout the Leaf. 

— In one class of leaves the vascular bundles entering the leaf 
are all gathered into the midrib, whence branches run into all 



n8 



CIRCULATION OF WATER AND SOIL SOLUTES 



parts of the blade. These are known as netted-veined leaves 
(Fig. 58, A). In another class all of the bundles are not 
merged with the mid-rib, but some run an independent course 
from base to apex, as seen in grass leaves. These leaves are 
called parallel-veined (Fig. 58, B). In the netted-veined sort 
the veins divide and subdivide until the meshes are extremely 
small — in some leaves approximately 0.2 mm. in diameter, and 
the ultimate branches end free in the mesophyll. In the par- 
allel-veined type the main veins running from base to apex 
are united by frequent cross branches (Fig. 58, D). Where 
the ultimate branches are 0.2 mm. apart water from them needs 




Fig. 59. Semi-diagrammatic cross-section of a leaf showing by arrows how the 
water passes from the tracheal elements of a vein into the border parenchyma cells, 
and thence into the palisade and spongy parenchyma, from which it evaporates into 
the intercellular spaces and passes from the leaf through the stomata. a, upper 
epidermis; b, lower epidermis; c, palisade parenchyma; g, spongy parenchyma; d, 
border parenchyma; e, tracheal elements; and the stippled cells below e, the phloem 
cells. 



to flow laterally only 0.1 mm. to supply the mesophyll cells 
at the center of the mesh. Illustrations of this kind make 
clear the general fact that the provision for the distribution 
of water throughout the leaf is very efficiently wrought out. 
In the successively smaller branches of the bundles in leaves 
we find the tracheal elements becoming fewer and srrialler 
until the smallest ramifications may have but a single line of 
tracheids. The phloem elements also are successively reduced 



POWER CONCERNED IN ASCENT OF WATER I 1 9 

in the smaller and smaller branches ; the sieve tubes give way 
to undivided mother cells of sieve tubes (see page 38) and 
companion cells, and these finally are succeeded by border 
parenchyma cells merely. Thus it happens that at the ends 
of the ultimate branches the vascular bundles may be repre- 
sented by a single tracheid merely since the border paren- 
chvma is not morphologically a part 'of the vascular bundle. 

\Yater is drawn by osmosis from the tracheids by the bor- 
der parenchyma cells, and from these in the same way by the 
palisade and spongy parenchyma. From these tissues it is 
for the greater part evaporated into the intercellular spaces, 
and passes thence through the stomata into the external atmos- 
phere (Fig. 59). In this way some plants give off in twenty- 
four hours of a hot summer day as much as 10 c.c. of water 
for each square centimeter of leaf surface; but the average 
rate is much lower. A large birch tree has been found to 
give off from 300 to 400 kilograms of water in twenty- four 
hours. 

Only a relatively small part of the water taken into the 
palisade and spongy parenchyma cells is used in the manufac- 
ture of carbohydrates. (See page 144.) 

The Power Concerned in the Ascent of Water. — The 
force of osmosis in the root hairs and cortical cells of the 
young roots sets up the flow of water from the soil into the 
tracheal system; and this force communicated to the tracheal 
elements is at times sufficient to raise water many feet; but 
that it does not work fast enough to be the dominant force 
when transpiration is at its height is shown by the fact that 
at such times there is a negative pressure in the water high- 
ways, which may be taken as an indication that the effective 
power is in the form of a pull from above. 

The tracheal tubes and tracheids are small enough for capil- 

' larity to be a significant force in water ascent and in sustaining 

the weight of the water columns; and doubtless capillarity 

does play an important part. But it seems from the data now 

available that capillarity under the conditions existing in plants 



120 



CIRCULATION OF WATER AND SOIL SOLUTES 



can not lift the water fast enough, nor high enough to supply 

the taller trees. 

It seems that the palisade and spongy cells in the leaves 

must be exercising a powerful 
osmotic pull on the water in the 
tracheal elements in the veins; 
and this pull communicated 
downward might explain the 
negative pressure in the water 
highways when transpiration is 
rapidly going on. 

Although the medullary rays 
and wood parenchyma are in 
direct contact with the tracheal 
elements and take water from 
them (Fig. 60), it is now very 
doubtful whether the living cells 
in the rays and wood paren- 
chyma are essential factors in 
water ascent, for many liters 
of water have been drawn up 
through the trunk of a tree after 
the tissues in the trunk have 
been killed by poisonous solu- 
tions to the height of sixty feet. 
Again, when a branch with 
foliage is placed in a solution of 
indigo-carmine, a stain which 
does not at all enter living cells, 
the dye is found nevertheless 
to rise in the tracheal elements 
along- with the water; and it 

tic 60. Diagram to show the path » » 

of the water as it rises to, and escapes may be inferred from this that 

from, the leaves. ... ., 

the water holding the dye in 
solution has risen without being drawn into, and forced 
along by, living cells. Osmotic intake by the roots, capil- 




PATH OF WATER ASCENT 121 

larity. osmotic suction by the mesophyll cells of the leaves, 
acting- together, seem to be the chief forces concerned in water 
ascent. 

Path of Water Ascent. — The path of the ascent of water 
is clearly mapped out by placing a leafy stem in a weak solu- 
tion of eosin or indigo-carmine. If the stem is then studied 
by means of longitudinal and cross-sections before the stain 
has had time to diffuse from the tissues through which it is 
rising into surrounding tissues, it will be found that only the 
tracheal tubes and tracheids are stained; and when the stain 
has risen into the leaves it is the tracheids in the veins that 
first show its presence. Furthermore, if the cut end of a stem 
having foliage be dipped into melted gelatine or paraffin the 
tracheal tubes will be plugged up for some distance, so that 
the end of the stem can be trimmed to expose all the tissues 
without removing the plugs from the tracheal elements. If 
the end of the stem is then submerged in water the leaves soon 
wither, while in a control experiment employing a similar 
branch with the tracheal elements left open the leaves continue 
fresh and unwilted. These and other experiments leading to 
similar results leave no ground for doubt that the path of water 
ascent from the roots is through the cavities of the tracheal 
tubes and tracheids. 

Influence of Environment on the Water Conducting 
Tissues. — The amount of tissue devoted to the circulation of 
water depends upon the intensity of the demand for water 
to supply the loss by transpiration. In submerged water plants 
where transpiration does not take place the tracheal elements 
are hardly more than vestigial. In water plants with foliage 
borne above the surface the water-conducting tissues are better 
developed, while in land plants these tissues spring into still 
greater prominence. The amplitude of water-conducting tis- 
sues reaches its climax in tall-growing climbing plants with 
slender stems and large crowns of foliage, where the distance 
to be traveled is far and the demand for water through trans- 
piration large ; and in those trees also, such as the willows and 



122 



CIRCULATION OF WATER AND SOIL SOLUTES 



Liriodendron (yellow poplar of commerce) whose roots find 
abundant water in the moist soil of intervales and along 
streams, and whose foliage is lavish in transpiration (Fig. 61). 




<£&>, 

<% 




o 



oo o 

CD 



c 



P 



Oq 



n 



o 

D 



Fig. 6 i. Camera-lucida drawings of equal areas of cross-sections of stems of 
A, hop; B, yellow poplar; C, water cress, and D, Psidum Galapageium which grows 
in a perpetually dense fog. The first two stems have need to carry relatively large 
quantities of water, and the last two relatively little. Only the tracheal tissues are 
here outlined. 

Illustrative Studies 

i. Study longitudinal radial sections of old Aristolochia 
stems made into permanent mounts and double-stained in ery- 
throsin and iodine green or in safranin and hematoxylin ; or 
if fresh sections are used mount them in aniline sulphate. 
Note the spiral and annular tubes next to the pith that have 
been formed by the procambium, and the different pitted kinds 
that have been formed by the cambium farther out. Draw 



ILLUSTRATIVE STUDIES I 23 

the different kinds and let the drawings of the tangential 
walls, namely, those that are on edge in the radial section, 
show clearly the thick and thin places in the walls — the thick 
places to keep the' tubes from collapsing and the thin places 
to allow the inflow and outflow of water and solutes. 

2. Study cross and longitudinal radial and tangential sec- 
tions of pine wood, double stained and permanently mounted. 
Study the cross-section first and draw to scale a small portion 
at the juncture of the early growth of one year with the late 
growth of the preceding year where a medullary ray cuts 
through. Look sharply for any means of interchange of 
materials between early growth, late growth, and medullary 
rays. If the drawing is not made as exactly as possible im- 
portant details will be left out. 

Now study the longitudinal radial section at the juncture of 
two rings of growth where a medullary ray runs across. Make 
a careful drawing to the same scale as that of the previous 
drawing. After studying the cross-section the appearance of 
the radial section is surprising. What we now want to do is 
to discover in one section every thing we have found in the 
other. To do this it is necessary to recognize in the radial 
section both the tangential and radial walls of the tracheids. 
In the cross-section these are easily recognized, since the radial 
walls run parallel with the medullary rays and the tangential 
walls are perpendicular to the rays. Notice that in the radial 
section the tangential walls are seen on' edge and the radial 
walls are lying flat. Every thing seen in the radial section can 
be found in the cross-section and vice versa but from different 
points of view and having a different appearance, and the same 
thing is true of the tangential section. Discover whatever 
means of communication there is between the tracheids later- 
ally and longitudinally and between the ray cells and the tra- 
cheids and between the two rings of growth. Are the medul- 
lary ray cells all alike ? 

3. Study the longitudinal tangential section and discover 
from this point of view all details that have been found in the 



124 CIRCULATION OF WATER AND SOIL SOLUTES 

other two. Look for provision for the flow of materials in 
all directions. Draw to scale a small portion where a medul- 
lary ray and tracheids join. Determine the number of med- 
ullary rays in one sq. mm. Discover whether every tracheid 
is touched once or more than once throughout its length by 
a ray. 

4. Criticize Fig. 52, which is constructed from the three sec- 
tions above studied. 

5. Study cross and longitudinal radial and tangential sec- 
tions of oak, and yellow poplar, double-stained and permanently 
mounted. Note by comparison of all sections what provision 
has been made for the interchange of materials between the 
tracheal tubes, tracheids, medullary rays, wood parenchyma 
and wood fibers. 

In the tangential sections determine how many rays occur in 
one sq. mm. and how many rays touch a tracheal tube for every 
mm. of its ascent. The purpose of this is to find out how com- 
plete is the provision for taking up and distributing the water 
and solutes carried vertically by the tracheal tubes. 

6. Compare for frequency and water-carrying capacity the 
tracheal elements in cross-sections of stems of oak, hop and 
water lily. Make diagrams to illustrate what is found. 

7. Study the tracheal tissues in leaves. Clear a leaf by 
boiling it in alcohol, placing it in 5 per cent, hydrochloric acid 
for about ten hours or over night, and keeping it for a while 
in a saturated chloral hydrate solution. Mount the leaf in 
chloral hydrate. The leaf should now be perfectly clear. 
Study it with low and high powers. Make drawings of the 
endings of the tracheal tissues of the veins. Measure the dis- 
tance apart of the ultimate branches of the veins. Determine 
by slow focusing from one surface to the other what position 
the tracheal tissues occupy in the leaf. We are in this way 
finding evidence of the efficiency of the tracheal elements in 
distributing water quickly to all cells of the leaf. 

8. Select a plant having leaves with prominent veins. Cut 
some of the veins across clear through. Does the leaf above 



ILLUSTRATIVE STUDIES I 25 

the cut wilt? Cut across more and more veins. How do you 
account for the results ? 

9. To map out the course of water ascent in a very vivid 
way place a young shoot of balsam or Ricinus into a weak 
solution of eosin. If the plants have been crowded in the seed- 
bed so that the stems are spindling and colorless, all the better 
for this experiment. After a while in the balsam stems the 
stain can be seen from the outside ascending the vascular bun- 
dles, and later extending into all the fine ramifications of the 
veins. Cross and longitudinal sections of the stem now show 
that the tracheal elements alone are stained, and a leaf cleared 
in 5 per cent, hydrochloric acid followed by saturated chloral 
hydrate will show the tracheal elements stained. Of course 
if the stems have been left long in the eosin this will diffuse 
from the tracheal to the surrounding tissues. 



CHAPTER VIII 
INTAKE AND CIRCULATION OF GASES 

Oxygen and Carbon Dioxide Necessary to Plants. — All 

plants liberate energy stored in complex compounds, and all 
of the higher plants and most of the lower need the free oxy- 
gen of the atmosphere to do this fast enough for their normal 
functions. The union of free oxygen with the protoplasm 
and reserve foods whereby these substances are broken down, 
and in consequence energy is liberated within the cells, is 
the essential process of aerobic respiration, while in anaerobic 
respiration the breaking down process releases energy without' 
the assistance of oxygen. This energy is used in keeping up 
the vital processes. Respiration, in other words, is the means 
of converting stored or potential energy into active or kinetic 
energy. This is the essential function of respiration without 
which plants could not live any more than animals could. Each 
living cell of the plant body must respire for itself, for the en- 
ergy liberated in one cell is not available in the next. We are 
led to conclude, indeed, that any part of the living protoplasm 
that is to benefit by the kinetic energy must take part in its lib- 
eration and be directly acted upon by the kinetic energy as it 
is being transformed from the potential condition. In a large 
plant body, therefore, consisting of masses of cells, there must 
be provision for access of oxygen to every cell, and for the 
elimination of carbon dioxide resulting from respiration. The 
oxygen absorbed by one cell may diffuse into one adjoining, 
and so on for a short distance ; but the surest way of securing 
to each cell all of the oxygen that its greatest demands may 
require is to have it exposed for a part of its surface to an air- 
carrying intercellular space, a condition that is approximated 
by an elaborate system of intercellular spaces. 

The cutinized and suberized walls of epidermis and cork, 
which, as we have learned, in Chapter IV, are necessary to 

126 



THE STOMATA I 27 

keep plants from drying up, also retard the inflow and outflow 
of gases, and this fact has made necessary the passage ways 
through the epidermis and cork known as stomata and lenticels. 

While every living cell in the plant body must have free 
oxygen for its respiration, all cells which make the food of the 
plant from carbon-dioxide and water, namely the green cells, 
and particularly those in the leaves, must have carbon dioxide 
brought to them. There are times in the spring and summer, 
during rapid growth or the storage of reserve foods in seeds, 
tubers, etc., when carbon dioxide must be absorbed in large 
quantities. But this gas exists in the atmosphere in very 
dilute solution, namely, there are between three and four parts 
of it in 10,000 parts of atmosphere, and every facility must 
be offered for its entrance and circulation. Accordingly we 
find stomatal openings through the epidermis of leaves, and 
relatively large, continuous, open ways between the food 
manufacturing cells within the leaves. 

Doubtless the need of the absorption and circulation of 
oxygen and carbon dioxide is the chief reason for the existence 
of an aerating system composed of openings and passage ways ; 
but vapor of water is excreted into the same channels and 
through them is thrown off into the air. How much this is 
merely incident to the fact that the passage ways are there, 
and how much the well-being of the plant demands this excre- 
tion of water has not been definitely determined. There are 
times of great turgidity of the tissues when water filtrates into 
the intercellular spaces ; but this water soon evaporates through 
the stomata and lenticels, leaving the spaces again open. 

The intercellular spaces, then, serve three main purposes : 
They provide for the admission and circulation of oxygen used 
in respiration, and of carbon dioxide employed in food-making, 
and they furnish a way for the elimination of water vapor. 
Other kinds of intercellular spaces and other uses for them will 
be spoken of in Chapter XII. 

The Stomata. — The stomata are minute openings, invisible 
to the naked eye, through the epidermis. They may be found 



128 



INTAKE AND CIRCULATION OF GASES 



on any part of plants except the roots, and they occur in great- 
est numbers in the leaves, where they average ioo to 300 per 

square millimeter ; and some- 
times, as in species of Olea 
and Brassica, they become 
as numerous as 600 to 700 
per square millimeter. 

A stoma is guarded by two 
guard cells (Fig. 62), which, 
as a rule, have the power 
automatically to open and 
close the stoma. The for- 
mation of a stoma comes 
about by the division of a 
protodermal mother cell into 




Fig. 62. A typical stoma in cross- 
section and surface view combined, k, 
guard cell; j, the gap or stoma between 
the guard cell; I, epidermal cell bordering 
a guard cell. 



two daughter cells and the 
dissolution of the middle la- 
mella of the walls separating 
these, thus forming a crevice, 
which may be closed, or 
opened out by the action of 
the guard cells to a breadth 
of approximately .008 mm., 
or about one nineteenth the 
thickness of this page (Fig. 
63). So minute are these 
clefts in the epidermis, even 
in their wide-open condition, 
that their value as entrance 
ways for carbon dioxide gas 
cannot be appreciated with- 
out experimental data. This, 
fortunately, is at hand. It 
has been found in purely 

physical experiments that this gas will diffuse at a faster rate 
through very minute openings in a membrane than through 




'^ 



Fig. 63. A, diagram showing rela- 
tive position of the guard cells in cross- 
section in the open and closed positions; 
the heavier line indicates the open posi- 
tion. B and C, early stages in the for- 
mation of stomata; at s, mother cells of 
guard cells are shown. D, s, s, two 
guard cells formed by the division of a 
mother cell. (After Sachs.) 



THE STOMATA I 29 

larger openings; and by having the minute openings frequent 
enough it is possible for it to pass through them as rapidly as 
if no membrane whatever were interposed. Physiological 
experiments show further that when the stomata are closed 
carbon dioxide does not enter the leaf rapidly enough for food- 
making. (See Chapter IX.) 

A better conception of the frequency of 
the stomata will be gained by a comparison HHiillll a 

of a and b, Fig. 64. a, which is a square a b 

of ; nam. on the side, contains 100 dots. fig. 6 4 . Diagram 

_.-.-. . , , r indicating the fre- 

Imagine this figure reduced to the size 01 quency of the sto- 
h. while retaining the same number of dots, ™ at * m a ^ as ^ are of 

o ' s mm. on the side 

and it will be conceived how small and containing 100 dots; 

., , 1 1 ,1 tIiese would have to 

numerous the stomata must be when there be crow ded into &, 
are 100 of them in a square millimeter. to approximate the 

average frequency of 

Then conceive of 300 or even 700 of them stomata in leaves. 

in the same area! In this way our idea 

of the size and frequency of the stomata becomes somewhat 

definite. 

The chief use of the stomata is to allow carbon dioxide gas 
to enter by diffusion from its very dilute solution in the atmos- 
phere. Oxygen also enters through the stomata, but so far as 
this gas is concerned they seem to be unnecessary, for respira- 
tion in which oxygen is employed seems to go on perfectly 
well when the stomata are closed or artificially plugged with 
vaseline, etc. Respiration can still take place under such cir- 
cumstances because there are 20 parts of oxygen in 100 parts 
of atmosphere, or about 500 times as much oxygen as carbon 
dioxide. 

Assuming that the chief function of the stomata is to admit 
carbon dioxide for food-manufacture, it is evident that the 
conditions which most favor food construction should also 
induce the opening of the stomata ; and this is found to be the 
case, for when the cells are turgid and the sun is shining — two 
conditions essential to the manufacture of food by the chloro- 
plasts in the leaves — the stomata stand open, while in darkness 
10 



130 



INTAKE AND CIRCULATION OF GASES 




and when turgidity is low in the leaves, they close. Their 
behavior under these conditions admits of a simple physical 
explanation: The £uard cells contain chloroplasts (see Chap- 
ter IX), which manufacture sugar, etc., when the sun is shin- 
ing. These products become dissolved in the cell sap and in- 
crease its osmotic pressure. Water is then drawn osmotically 
into the guard cells from the surrounding epidermal cells until 
the former become turgid and swollen, and 
in this -condition the guard cells draw apart 
and leave a passage way between them into 
the interior of the leaf. This behavior is 
apparently due to the fact that the wall of 
the guard cell farthest from the opening is 
more extensible than the wall bordering the 
opening and bulges out farther when the 
turgidity of the cell increases, and drags 
the wall bordering the opening along with 
it (Fig. 63, A). This action is made clear 
by a simple experiment with rubber tubing. 
Two short pieces of tubing are connected 
by a Y-tube with a water faucet. The 
lower ends of the pieces of tubing have been 
plugged and fastened together, and the ex- 
terior side of each piece has been pared thin 
with a sharp knife or scissors. When the 
water pressure is turned on the thin outer 
sides bulge and the pieces of tubing draw 
apart as shown by the dotted lines in Fig. 

65- 

Under exposure to light there may be 
other influences on the action of the guard 
cells than the increase in them of osmotic substances; but 
experiments have shown that the stomata do not as a rule 
open in an atmosphere devoid of carbon dioxide, and this 
points to photosynthesis (see page 144) in the guard cells, 
yielding substances that increase the osmotic pressure, as 



> 






/ 




1 


/ 




\ 


/ 




1 


ti 


| 




i 


1 




\ 






\ 






V 






\ 




1 


\ 


*i 


t 


\ 


[ 






i- 


-1 




LL 


JJ 



J I 



Fig. 65. Diagram 
of apparatus showing 
how the guard cells 
draw apart; /, j, posi- 
tion of the rubber 
tubing when the 
water pressure is 
turned on. 



THE STOMATA 13 I 

the main factor in stomatal action. It must be stated, how- 
ever, that experiments of this sort have given apparently 
contradictory results, for in some experiments with well- 
nourished plants in an atmosphere free from carbon dioxide 
the stomata have still been found to open on exposure to the 
light. 

We have noted the necessity of stomata for the admission 
of carbon dioxide for food construction, and the fact that in 
most cases they are not needed to let in oxygen for respiration. 
Are they a necessity in the elimination of water or transpira- 
tion? This process of course hastens the flow of water from 
roots to leaves, and soil solutes are swept along in this current, 
and the intake of the solutes is consequently hastened. It 
seems possible that in tall plants at least the passage of solutes 
from the roots to the crown by diffusion alone would be too 
slow for the needs of nutrition, and the stomata are doubtless 
very useful in this. But it may also be true that while the 
stomata are standing open to let in carbon dioxide during the 
hours of daylight a great deal more water incidentally passes 
through them than is needed for the ascent of solutes. In- 
deed, when the water supply in the soil is running low its trans- 
piration through the stomata becomes a real danger to the 
plant, and the ability to close the stomata when the guard cells 
lose their turgidity, as they would under such conditions, 
becomes a safeguard against harm and even death from dry- 
ing up. 

Even in bright sunlight the stomata will close when water 
evaporates through the guard cells faster than it can be taken 
in. The sensitiveness of the guard cells to variations in the 
water supply is shown when a plant with open stomata is taken 
from the moist air of a greenhouse to the dryer air out of 
doors ; then the stomata close in spite of the fact that the plant 
may be in bright sunlight. Some plants, such as the willows 
and alders, are found to have lost the power of closing the 
stomata, and the lack of this check on the transpiration stream 
may be the reason for the restriction of these plants to habitats 



132 



INTAKE AND CIRCULATION OF GASES 



where the roots find a perennial supply of water along streams 
and in wet soil. 

The stomata as a rule occur on both sides of leaves, but with 
greater frequency on the lower side. There are instances, as 
in the rubber leaf, and some oaks, plums and apples, where 

the stomata are all on the lower 
side, and still fewer instances, 
as in the water lily, where the 
stomata are practically all on the 
upper side. Where the leaves 
hang downward, like those of 
some poplars, or stand upright, 
as in the grasses, the number of 
stomata is about equal on both 
sides. 

Often when the stomata occur 
on one side only they make up 
in frequency there for the lack 
on the other side. Thus, per 
square millimeter of surface, 
Nymphaea alba has 460 stomata 
on the upper side and none on 
the under; Pirus malus has none 
on the upper side and 246 on the 
lower; while Triticum sativum 
has 47 on the upper and 32 on 
the lower side. 

The Relation of Stomata 
to the Environment. — In desert 
regions and in places where plants at certain seasons are in 
danger of suffering from scarcity of water it has been found 
advantageous to plants to equip the stomatal apparatus with 
devices that retard evaporation while allowing the diffusion of 
gases into the leaf. A common plan is to sink the stomata 
below the level of the general epidermis, so that each is at the 
bottom of a pit where the winds cannot sweep away at once 




Fig. 66. A, depressed stoma of 
the under side of a leaf of Am- 
herstia nobilis. B, depressed stoma 
of Hakea suaveolens. g, strands be- 
neath the guard cells; d, outer, and 
e inner, cavities. (After Haber- 
landt.) 



THE LENTICELS 133 

the water vapor as fast as it is transpired. Sometimes the 
outer wall of the guard cells is elevated in the form of a crater, 
or an elevated border of wax may serve to maintain a quiet 
atmosphere just above the stoma. However the pit or crater 
is produced it is sometimes made more effective by outgrowths 
that partially roof it over, as in Fig. 66. 

The Lenticels. — In stems where the epidermis is being 
replaced by cork the phellogen or cork cambium (see page 56) 
instead of producing ordinary cork tissue immediately below 
the stomata forms loose layers of cells, through the intercel- 
lular spaces of which gases pass in and out (Fig. 22). These 
groups of loose cells are called lenticels. As the lenticels be- 
come larger by additions from the phellogen they press out 
and burst the epidermis and appear at the surface as rounded, 
elliptical or oblong excrescences. 

The formation of lenticels outruns that of the cork tissue, 
so that when the cork has encased the stem and replaced the 
epidermis as a protective tissue the lenticels have taken the 
place of the stomata and have provided aeriferous channels 
from the exterior entirely through the periderm. (See page 

S7-) 

The intercellular spaces of the lenticels are in direct com- 
munication with the main intercellular channels of the bark 
and wood. This is demonstrated by sealing one end of a cut- 
off stem, submerging the stem with the closed end downward, 
and forcing air into the upper end. Air bubbles then issue 
from the lenticels. And when a ring of bark is removed from 
the upper end so that the air pressure tube is connected with 
the wood only the bubbles issue as before, showing that the 
lenticels communicate with the intercellular spaces in the wood 
as well as with those of the bark. Plainly the lenticels are 
for the aeration of the stem throughout its entire body. 

The radial distribution of gases that enter through the len- 
ticels is carried on through intercellular spaces running through 
the medullary rays, and from these the vertical distribution 



134 



INTAKE AND CIRCULATION OF GASES 



takes place largely through spaces between the cells of the 
xylem parenchyma (Fig. 6j). 

The main use of the lenticels is evidently for the interchange 

of gases in respiration, namely, 
for the intake of oxygen and 
the excretion of carbon diox- 
ide, since they occur on stems 
where respiration must be go- 
ing on continuously, but where 
the converse process of taking 
in carbon dioxide for food 
building is taking place to a 
very subordinate degree, if at 
all. Incidentally some vapor of 
water passes out through the 
lenticels, but that they are need- 
ed for this seems improbable. 
The Intercellular Spaces. — 
If there were no intercellular 
spaces threading the plant body 
in all directions the cells lying 
remote from the surface would 
have access to the gases of the 
atmosphere only as they came 
by slow diffusion in solution in 
the cell sap, and this would be 
inadequate for all cells that are 
actively living and working, 
such as those cells that construct 
and store, digest and otherwise 
transform food, and all cells 
that are passing through cell 
division, growth and differen- 
tiation. And so air spaces are formed between cells throughout 
the whole plant body from tips of roots to tips of branches 
and from stomata or lenticels to the pith. The plant is, in 




Fig. 67. Diagram suggestive of the 
distribution of intercellular spaces 
throughout a plant. The heavy hori- 
zontal lines in stem and root indicate 
intercellular spaces in medullary rays. 
The arrows indicate the entrance of 
gases through the stomata of the 
leaves and stomata and lenticels of the 
stem, and anywhere over the roots, 
which are less water-proofed than the 
leaves and stems. Lenticels have been 
found where the lateral roots grow 
out from the main root. 



THE INTERCELLULAR SPACES 135 

fact, riddled with these fine spaces to such an extent that prob- 
ably the cells farthest from them are distant not more than 
a few tenths of a millimeter. 

The formation of the intercellular spaces begins in the 
meristematic tissues before the differentiation of the perma- 
nent tissues has begun; and this should be expected since the 
dividing and growing cells of the meristems respire rapidly 
and make relatively large demands for oxygen and the elimi- 
nation of carbon-dioxide. 

The intercellular spaces are made by the dissolution of the 
middle lamella of the cell walls, particularly where three or 
four cells come together, and a subsequent growing apart of 
the cells at these places. Another mode of their formation 
occurs also where in some instances the spaces are of uncom- 
mon size, as in the hollow stems of grasses, thistles, Equi- 
setums, etc. In cases of this kind the large central cavity is 
formed by the breaking down and disappearance of the inte- 
rior fundamental tissue. The first method of intercellular 
space formation is called schizo genous and the last, lysigenous. 

As a rule the intercellular spaces are larger in leaves than 
in other parts of the plant body. The percent, of the volume 
of leaves occupied by intercellular spaces is different in dif- 
ferent plants, varying all the way from 71.8 per cent, in Pistia 
Texensis, a floating aquatic herb, to 3.5 per cent, in Begonia 
hydrocotyli folia, a succulent creeping herb native to Mexico. 

A large volume of intercellular space in leaves is a response 
to the need of them to provide the photosynthesizing or food- 
constructing cells (see page 146) with carbon-dioxide. Where 
the habitat provides plenty of water the demand for large 
spaces in the leaves can be satisfied without danger, as in 
Pistia ; but where water is hard to obtain and too great trans- 
piration might result from large intercellular spaces this pro- 
vision for the circulation and storage of carbon-dioxide must 
be sacrificed to a greater or less extent, as in the Begonia 
above mentioned, and in plants in general of desert and other 
xerophytic habitats. 



36 



INTAKE AND CIRCULATION OF GASES 



In the stems and roots of plants growing under mesophytic 
or average conditions of moisture the intercellular spaces are 
very minute and can be made out only under close scrutiny 
when good sections are prepared and studied under great 
magnification. It seems from this that the large percentage 
of oxygen in the atmosphere enables the gas to diffuse through 
the plant body fast enough without much space being given 
over to diffusion highways. The case is different, however, 
in water or marsh plants, that is, plants under hydrophytic 




Fig. 68. Low-power, camera-lucida drawings of A, cross-section of stem of Juncus; 
and B, stem of Nymphaea, showing large intercellular spaces at i. In A the black line's 
traversing the section are really chains of cells. 



conditions, where oxygen must be taken from its dilute solu- 
tion in water, or must travel longitudinally through the length 
of the plant from its place of intake in floating or aerial leaves, 
or from its place of liberation by photosynthesis in the green 
parts. In these plants the intercellular spaces become rela- 
tively very large, as in Nelumbo, Heteranthera, Juncus, etc. 
(Fig. 68). 

It seems that in mesophytic and xerophytic plants (meso- 
phytes and xerophytes or plants at home under average condi- 
tions of water supply, and where water is scarce, respectively) 
the intercellular spaces are so small that each part, root, stem, 
and leaves must take in air more or less independently of the 
others — the stem can not supply the root with air, neither can 
the root or the leaves supply the stem, although seedlings of 
Pisum and Vicia are known to be exceptions to this rule. If 



MOTIVE POWER IN DISTRIBUTION OF GASES 137 

the soil in which mesophytes or xerophytes are growing' be 
inundated, so that the stems and leaves are not submerged, 
the roots can not get air and the plants die. The same thing 
may happen when earth is filled in around trees to a depth of 
several feet. 

Diosmosis of Gases into and from Living Cells. — The 
wall of every living cell is infiltrated with water, and a very 
thin film of water covers its free surface. Therefore the 
gases in the intercellular spaces must, before entering the cell, 
go into solution in this water, and in this condition diosmose 
through the cell wall. Likewise gases that are to be given 
off from the cells must in solution diosmose through the cell 
wall and break away from the superficial film of water into 
the intercellular space or outer air. The dry walls of dead 
cells are much less permeable to gases than those of the living 
ones soaked with water. 

Motive Power in the Distribution of Gases throughout 
Plants. — Gases pass by diffusion in and out through the sto- 
mata and lenticels, and throughout the intercellular spaces. 
By this process the atmosphere does not move as a whole but 
each component gas moves independently in obedience to the 
laws of diffusion according to which the movement is from the 
place of greater concentration of a particular gas to that of 
less concentration. So that if carbon dioxide is being used 
in photosynthesis by any tissue, or oxygen is being employed 
in respiration, more of these gases will flow to that tissue by 
diffusion. Or if respiration is pouring carbon dioxide into 
the intercellular spaces, or photosynthesis is loading them with 
oxygen, these gases will flow towards the exterior and pour 
out through stomata and lenticels. Interchange of gases in 
this way is constant so long as their concentration inside and 
outside the plant body is different. In the daytime there 
would be a flow of oxygen from the photosynthetic tissues 
towards the other parts that are breathing, as well as towards 
the exterior, and a flow of carbon dioxide towards the photo- 
synthetic tissues from those that are giving it off in respira- 



I38 INTAKE AND CIRCULATION OF GASES 

tion, as well as from the exterior. In parts of plants desti- 
tute of chloroplasts, and hence incapable of photosynthesis, 
there would be no evolution of oxygen, but on the contrary 
there would be a continual production of carbon dioxide by 
respiration, and this gas would flow thence towards the photo- 
synthetic tissues and towards the exterior. When darkness 
falls no part can longer photosynthesize while all parts con- 
tinue to respire, and therefore a flow of oxygen more exclu- 
sively from the exterior to all parts sets in, and the return 
flow of carbon dioxide becomes more exclusively towards the 
exterior, since the photosynthetic tissues can no longer make 
use of it. Thus the flow of gases by diffusion throughout the 
plant body while in necessary obedience to physical laws is 
yet in direct accordance with physiological demands. 

Movements of the atmosphere en masse throughout the 
plant body are accomplished by differences between external 
and internal pressures resulting from fluctuations of tempera- 
ture within and without, and by compression and expansion 
of the intercellular spaces caused by swaying of the plant to 
and fro, and by fluctuations in the turgidity of the tissues as 
the ratio between absorption and transpiration rises and falls. 
Thus when the temperature of the plant body falls below that 
of the exterior the air in the intercellular spaces contracts and 
outside air flows in, and the reverse process takes place when 
the temperature of the plant rises above that of the outside 
atmosphere. Again, when transpiration is going on faster 
than absorption by the roots the cells lose in turgidity, con- 
tract, and leave more room for the intercellular spaces, and 
pressure from without forces more air in. When transpira- 
tion subsides following the overclouding or going down of the 
sun, or increase in the atmospheric humidity, the cells become 
more turgid and compress the intercellular spaces so that the 
air in them is in part forced out. The relatively rapid ex- 
changes of air brought about in these ways may be of decided 
value to the functions employing oxygen and carbon dioxide. 
Free nitrogen, not being employed by the higher plants, may 
here be left out of the discussion of gaseous interchange. 



illustrative studies 139 

Illustrative Studies 
i. Study the stomata of different leaves. Bleach and clear 
the leaves as described in Illustrative Studies, paragraph 7 of 
the last chapter. Study with both low and high powers. With 
objects so transparent as these it is best to illuminate with 
oblique light by setting the mirror-bar at an angle with the per- 
pendicular. Mount the leaf or a portion of it in chloral 
hydrate, first with the under side up. With the eyepiece mi- 
crometer, and using the low power objective, determine the 
number of stomata in one sq. mm. Measure the stomatal 
apparatus and the opening. Draw a stoma together with the 
surrounding epidermal cells, using the high power. Turn the 
leaf upper side up and determine the number of stomata as 
before. 

2. Study the stomata in leaf cross-sections. Sections cut 
free-hand will do. Mount several of the thinnest sections in 
glycerine and with the high power find a median section 
through a stoma. W^hat provision do you find for the distri- 
bution of gases after they have entered through the stoma? 
Compare the stoma with the diagram of Fig. 63, A. Would 
it probably work as there set forth? Draw the stoma and 
epidermal cells adjoining it and the intercellular spaces leading 
from it. 

3. Look for intercellular spaces in the permanent prepara- 
tions prepared for the previous chapters and make drawings 
and measurements of some of them. Do you find every cell 
somewhere touching an intercellular space ? 

4. Study intercellular spaces in the stems of water lily, 
Juncus, or other water and marsh plants. Permanent prepa- 
rations or free-hand sections mounted in dilute glycerine may 
be used. Draw some of the spaces by outlining the cells bor- 
dering them. Estimate the percentage of stem volume occu- 
pied by these spaces. 

To show that the intercellular spaces in the leaf communi- 
cate with those in the stem fill a bottle with water, push in a 
perforated cork so that its upper surface is below the mouth 



14° INTAKE AND CIRCULATION OF GASES 

level; cut off the shoot of a young Ricinus plant and insert the 
stem through the hole in the cork; remove all water from the 
top of the cork and pour over it melted rosin one part and 
beeswax one part, so that no air can enter the bottle except 
through the intercellular spaces of the plant; set the plant in 
the sun but shade the bottle. As water evaporates through 
the leaves air can be seen bubbling from the submerged end 
of the stem. Explain how this happens. 

5. Examine lenticels on any woody stem that shows them 
well, — elderberry, for instance. Study with a high power a 
thin cross-section of a lenticel and draw it. 

To show that the lenticels are in communication with the 
general intercellular system of the stem, connect one end of a 
stem by rubber tubing with a bicycle pump ; then submerge the 
whole stem in water and force air into it. Do bubbles come 
out through the lenticels ? Explain the result. 



CHAPTER IX 

CONSTRUCTION OF THE PLANT'S FOOD 

The Source and Uses of Food. — It is the business of the 
green plants, known also as the autophytes, or independent 
plants, to make the food that is consumed by themselves as 
well as by other plants and animals. This they do by com- 
pounding carbon, which they get from the carbon dioxide of 
the atmosphere, hydrogen and oxygen from water, and nitro- 
gen, phosphorus and sulphur from the soil, into sugars, 
starches, oils, proteids, and some other less common forms of 
food. These are the food of plants, made by the green plants 
for their own use, but consumed also by parasitic plants and 
by animals, all of which are directly or indirectly parasitic 
on plants. 

It is sometimes said that plants live on inorganic matter, 
animals on organic. This, however, is not an exact state- 
ment. The food of plants and animals is the same. The 
difference is, rather, in this, that green plants make their food 
from inorganic matter, namely, from carbon dioxide, etc., as 
above stated, while animals can not make their food, but must 
take that provided by plants. A bean plant, a corn plant, an 
oak plant, all green plants, so long as the leaves are green, are 
making food while the sun shines and furnishes them the 
energy with which to work. 

The food has two distinct uses : It provides materials for 
the construction of the body and energy for carrying on the 
vital functions. The living protoplasm and the cell wall, — 
every part of the plant body in all its details, are made from 
materials that first appeared as food in the form of sugar, 
starch, oil, proteid, etc., and practically everything that plants 
do, aside from food-construction where the sun supplies the 

141 



142 



CONSTRUCTION OF PLANT S FOOD 



needed energy directly, is accomplished by energy released in 
the decomposition of these foods or substances made from 
them. 

The green leaves, therefore, are the organs where the sun's 
energy is transformed so that it can be stored for use after- 
wards where and when it is needed. They are the organs 
where the materials of the inorganic world are assembled in 
such a form that they can be used in the construction of the 
living body. 

How energy is obtained in the decomposition of food will 
appear from a specific example: When a definite amount of 
sugar or starch is made within the leaf from carbon dioxide 
and water a certain amount of sun's energy is employed and 
transformed from the active or kinetic to the passive or poten- 
tial state. This energy is not destroyed or lost; it is simply 
changed from one condition to another. To illustrate : a cer- 
tain amount of energy is required to lift a brick from floor 

to table. The energy spent 
in doing this now rests quies- 
cent in the brick in virtue of 
its position above the floor, 
and reappears as active en- 
ergy when the brick falls. So 
when sugar or starch is trans- 
formed into carbon dioxide 
and water from which it was 
made the energy from the 
sun employed in compound- 
ing it is set free (comparable 
to the falling of the brick) 
and a part of it can then be 
used by the plant for other 
purposes, as in the construc- 
tion of proteids from carbohydrate, etc., the synthesis of pro- 
toplasm, the overcoming of resistance, and in other ways. 




Fig. 69. Cross-section of a portion 
of the blade of a leaf, showing upper 
epidermis at a, lower epidermis at b, pali- 
sade parenchyma at c, spongy parenchyma 
at d, and tracheids from the end of a 
vein at e. 



FOOD-BUILDING APPARATUS 



143 



The sun's energy made potential in sugar and the like is, by 
the circulation of these substances, distributed to all parts of 
the body where every living cell can make use of it, and where, 
if not immediately wanted, it can be stored for use at some 
future time. 

Food-Building Apparatus. — It is the special business of 
leaves to make the food, and there the palisade and spongy 
parenchyma are the tissues directly concerned in the work 
(Fig. 69), since the new food makes its appearance first in 
them. Each individual cell of these tissues is therefore a 
food-building unit, and the factors concerned in food synthesis 
can be followed out in seeing how one of these cells does its 
work, namely, how it obtains the raw materials and the sun's 
energy, how the arrested energy of the sun 
is utilized, how the finished product is dis- 
posed of, and how the apparatus of the cell 
is kept in working order. 

The Chloroplasts. — The chloroplasts 
(Fig. 70) are plastids that contain the green 
pigment chlorophyll (see page 309). They 
are the organs of the cell that are directly 
concerned in food-construction from car- 
bon dioxide and water. This is known 
from the fact that this process does not 
go on where chloroplasts are absent; and 
where starch is formed, as is nearly always 
the case, it invariably is found within the body of the chloro- 
plast. 

The chloroplasts and the green chlorophyll are distinct 
things, for alcohol will extract the chlorophyll and leave the 
chloroplasts as before but devoid of color. (See about origin 
of chloroplasts on page 9.) The chlorophyll is a pigment 
which the chloroplasts manufacture with the aid of the sun- 
light. The significance of the chlorophyll is that it arrests a 
part of the energy from the sun and transforms it in such a 
way that the chloroplasts can use it in food synthesis. We 




Fig. 70. Diagram- 
matic representation 
of a single palisade 
cell, with chloroplasts 
lining the walls. 



144 



CONSTRUCTION OF PLANTS FOOD 



Fig. 71. Starch grains from the palisade 
cells of a Euphorbia leaf, magnified about 1800 
diameters. 



can not state, and do not at all know, the details of this 
process. Clearly the chloroplast, a living body, does the work, 

but to do this it needs to 
be energized by the sun, 
and this is apparently 
what the chlorophyll is 
instrumental in bringing 
about. Because the sun- 
light furnishes the en- 
ergy for food construc- 
tion the process is called 
photosynthesis. 

The first visible food 
made by the chloroplasts 
is starch in the form of 
very minute grains ( Fig. 
71). There is reason 
to believe that sugar is formed before the starch appears, and 
presumably in the chloroplasts also, but it is soluble in the 
cell sap and probably does not long 
remain where it is first formed, 
but passes by diffusion from the 
chloroplasts into the cell sap, filling 
the cell cavity, and thence into the 
tissues devoted to food-conduction, 
as told in the next chapter. 

The minute size of the chloro- 
plasts affords them large surface in 
proportion to their volume, and this 
gives a great advantage in the ab- 
sorption of raw materials and en- 
ergy, and in the elimination of the 
finished product. 

The chloroplasts are always em- 
bedded in the cytoplasm close 
against the cell wall, and this peripheral position is appar- 




Fig. 72. Diagram to show 
the intake of carbon dioxide 
by the palisade cells from the 
intercellular spaces, the absorp- 
tion by the chloroplasts of 
water from the cell sap, and 
the passage of food from the 
chloroplasts into the cell sap. 
The palisade cells are shown in 
cross-section, as they would be 
if the leaf were cut parallel 
with the surface, namely, tan- 
gentially. 



THE SUNS ENERGY 



145 




ently an advantage to every phase of their work; for, as Fig. 
72 will show, on one side water is presented to the chloro- 
plasts from the cell sap, and on another carbon dioxide from 
the intercellular spaces, and the vacuole affords an unob- 
structed way for the removal of the manufactured product. 
Also by this arrangement the light entering the leaf from all 
parts of the sky has a clearer path to every chloroplast through- 
out the leaf than would otherwise be the case (Fig. 73). 

The Sun's Energy. — The amount 
of the sun's energy that comes to 
the earth every day is too vast for 
us to realize. A part of that which 
falls upon the leaves of plants is 
reflected, but the greater part enters 
and, since the epidermis is trans- 
parent, penetrates to the chloro- 
plasts. Fig. 74 shows in a diagram- 
matic way what happens to it there. 
It is a matter of common observa- 
tion that very little light penetrates 
through a leaf even in full sunlight ; 
and when the little that comes through is analyzed with a 
spectroscope it is found that it consists of some red, orange, 
and yellow, and a relatively large amount of green, while the 
blue and violet parts of the spectrum have been almost entirely 
absorbed by the leaf. Less than 2 per cent, of the absorbed 
light, we know from experiment, the chloroplasts employ in 
food-building, and approximately 98 per cent, is used in the 
evaporation of water. 

By a very ingenious method, known as Engelmann's bac- 
terium method, it is demonstrated that the red light rays are 
far more potent in photosynthesis than the others, although 
the orange and yellow rays are much employed in this work; 
while the green, blue, and violet rays are of all the least useful. 
The Engelmann demonstration, based on the fact that oxygen 



Fig. 73. Diagram showing how 
the position of the chloroplasts 
against the vertical walls of the 
palisade cells exposes them to 
good advantage to light from all 
quarters of the sky. 



146 



CONSTRUCTION OF PLANT S FOOD 



Light 




is evolved during photosynthesis, is carried out as follows: 
Motile bacteria that are known to be attracted by oxygen, 
together with a filamentous alga, are placed on a glass slip 
in a drop of water and a coverglass is 
placed on and sealed around the edges air- 
tight with vaseline. Examining the prepa- 
ration with a high power objective it is 
observed that when it is exposed to the 
light the bacteria flock together along the 
alga, showing that oxygen is being evolved 
by it. If the light is now screened off 
the bacteria scatter, only to assemble along 
the alga again immediately the screen is 
removed. This shows that photosynthesis 
begins at once in the light and ceases as 
suddenly in darkness. After these prelimi- 
nary observations a small spectroscope 
adapted to the purpose is placed beneath 
the microscope stage. Light is thrown by 
the mirror through the spectroscope and the 
preparation is so adjusted that the colors 
of the resulting minute spectrum succeed each other along 
the length of the alga. It is then seen that the bacteria flock 
about the alga where it is illumined by the red light, gradually 
thinning out in the orange and yellow until relatively very 
few hover in "the regions of the green, blue, and violet. There 
seems to be no other inference from this than that the food- 
constructing or photosynthetic efficiency of each part of the 
spectrum is measured by the relative number of bacteria col- 
lecting in its domain. Fig. 75 shows how the red portion 
surpasses the others in this respect. It has been found that 
light that has passed through one leaf is too weak in the red 
to energize the chloroplasts in another. 

The Palisade Cell the Chief Photosynthetic Unit.— Most 
of the work of food-construction is done in the palisade cells, 
since they occupy the upper half of the leaf where the incom- 



Fig. 74. Diagram 
to show the activi- 
ties going on in a 
palisade cell. The 
arrows from the 
chloroplasts into the 
cell cavity indicate 
movement of photo- 
synthesized product. 
The thicknesses of 
the arrow shafts un- 
der " Light " indi- 
cate the relative light 
intensities. 



THE PALISADE CELL 



H7 



ing light is strongest, and they have four to five times as 
many chloroplasts as the cells of the spongy parenchyma have. 
When food synthesis is going on a palisade cell is the scene 
of great activity. Fig. 74 is a diagrammatic expression of 
this fact. Here for simplicity a single chloroplast in a pali- 
sade cell is made to represent the many smaller ones that 
actually occur. Light penetrates the chloroplast from above, 
carbon dioxide flows in from the intercellular spaces, and 




Fig. 75. Diagram to show the effect of different portions of the spectrum on 
photosynthesis, a to F, different regions of the spectrum from red to blue. A fila- 
mentous alga lies across these, and bacteria are collecting about the alga, with greatest 
frequency in the red between B and C, indicating the greatest evolution of oxygen 
there. (After Pfeffer.) 



water enters from the vein below the cell. The food product 
in the form of sugar diffuses from the chloroplast into the 
cell sap and thence into the food-conducting cells in the vein. 
Oxygen as a by-product of food-construction diffuses into the 
intercellular spaces, and into them water also evaporates from 
the cell sap. This stream of activities is none the less real 
because noiseless and unseen by the eye. 

The steps in the chemical process of food-construction can 
not be followed, but it is interesting to note how simple the 
process might be, as, for example, 6C0 2 + 6H 2 = C 6 H 12 6 
(glucose) -f- 60 2 . 

Relation of Leaf as a Whole to Photosynthesis. — A leaf 
from the standpoint of its cellular anatomy consists of three 
distinct parts : the epidermis, the ground or fundamental paren- 



148 



CONSTRUCTION OF PLANT S FOOD 



chyma, and the vascular bundles (Fig. 76). The epidermis 
has a waterproofed outer wall after the manner of a typical 
epidermis (see page 29), and it is perforated on one or both 



Epidermis 




Epidermis 
Fig. 76. Diagram to show the three general tissue regions of a leaf. 

surfaces of the leaf with stomata to allow the entrance of 
carbon dioxide as described in Chapter VIII. The ground 
parenchyma, also called the mesophyll, is differentiated into 
three kinds of tissues: the palisade parenchyma, occupying 
the upper half; the spongy parenchyma, of the lower half; 
and the border parenchyma, forming a sheath around the 
vascular bundles in the veins (Fig. 78). The border paren- 
chyma cells deliver water from the veins to the palisade and 
spongy parenchyma, and they gather food from these and 
conduct it to the sieve tubes in the larger veins, as described 
in the next chapter. 

The intercellular spaces in the spongy parenchyma are much 
larger than those in the palisade parenchyma, and they freely 
communicate with one another and with the intercellular 
spaces in the palisade parenchyma, and so are well suited to 
receive and distribute the carbon dioxide which enters mostly 
through the under surface because most of the stomata are 
there as a rule. The intercellular spaces in the palisade paren- 
chyma are not easily made out in cross-sections of leaves, but 
in sections parallel to the leaf surface (tangential sections), 



RELATION OF LEAF TO PHOTOSYNTHESIS I49 

or when looking through a bleached leaf with a microscope, 
they appear as in Fig. jj, where every palisade cell borders on 
one or more of them for a part of its surface. This general 
view of leaf anatomy shows how all the parts are related in 
the interest of photosynthesis. 

It will be well now to recapitulate briefly the main facts in 
what has thus far been told about the leaf : The epidermis is 
transparent and lets the light through. The chloroplasts in 




Fig. tj. Showing intercellular spaces: f, between the palisade cells; e, in a leaf; 
g, border parenchyma; h, tracheal elements of the vein. Camera-lucida drawing of 
a tangential section of a leaf. 

the palisade cells absorb most of the light and use approxi- 
mately 2 per cent, of its energy in carrying on food synthesis. 
Light that escapes through the palisade parenchyma is arrested 
so completely by the spongy tissue that not enough goes on 
through the leaf to be useful to other leaves. The intercel- 
lular spaces of the spongy parenchyma receive and distribute 
to all parts of the leaf the carbon dioxide that has entered 
through the lower surface. The border parenchyma cells 
deliver water from the veins to the rest of the mesophyll, and 
receive food from palisade and spongy cells and together with 
the phloem part of the veins transport it out of the leaf. The 



ISO 



CONSTRUCTION OF PLANTS FOOD 



conditions necessary to photosynthesis are these: The photo- 
synthetic apparatus must be present in working- order; there 
must be light; the stomata must stand open and admit C0 2 ; 
the veins must bring water; the veins must carry away the 
food or its accumulation will hinder its further construction. 

Fig. 78 represents in a general way the cellular architecture 
of a leaf. The leaves of different plants of course vary from 
this in certain details. 

In the leaf of the India rubber tree, Ficus elastica (Fig. 
79), at the upper surface the epidermis is succeeded by a clear 




Fig. 78. Diagram to show the architecture of a typical leaf in the region of one 
of the lateral veins. The shaded parts amongst the palisade and spongy parenchyma 
represent intercellular spaces. 



tissue two to three cell layers deep which serves as a water- 
storage tissue. Then follow two to four layers of palisade 
cells, several layers of spongy cells, two to three layers of rela- 
tively small water-storage cells, and last the lower epidermis. 
In the spaces between the veins where the palisade cells can not 
communicate directly with the food-conducting cells the spongy 



RELATION OF LEAF TO PHOTOSYNTHESIS 



I^I 



rage tissue 




cells collect the products of the palisade cells and deliver them 
to the veins by the indirect route shown in Fig-. 79. This 
device is by no means peculiar to the rubber leaf, but is very 
generally employed. 

In Indian corn each of the vein- 
lets is surrounded by a sheath of 
border parenchyma cells, and these 
in turn by palisade cells radiating 
from them, as shown in Fig. 80. 
In corn, as indeed in many grasses, 
starch is not formed in the pali- 
sade cells at all, but the paren- 
chyma sheath on bright days is 
well filled with it (Fig. 80). Of 
course the palisade cells manufac- 
ture soluble carbohydrate abun- 
dantly, and the starch in the 

Sheath Cells doubtless represents Fig. 79. Cross-section tough 

J- a portion or rubber leaf, snowing 

a SUrpltlS that COmeS tO the the large percentage of water- 

, ., in r ... ,. , storage tissues on both sides of 

sheath cells from the palisade tis- the leaf> and the relation of the 
sue faster than it can be conducted P alisade and spongy parenchyma 

to the lateral veins. 

away. 

In the Agave, Codonanthe, etc., the leaves are very thick 
<j and palisade parenchyma sometimes 
stands against the epidermis on 
both sides, while the mesophyll 
cells making up the bulk of the 
leaf serve mainly as water-storage 
cells (Fig. 81). Leaves of this 
sort with large amounts of water 
stored against time of need are not 
infrequent in desert regions or 
where a rainy season is succeeded 
by a dry one. 

Conditions Affecting Photosyn- 
thesis. Light. — The amount of 




Fig. 80. Cross-section of a 
portion of a leaf of Indian 
corn, a, upper, and b, lower 
epidermis; c, c, palisade cells; 
n, border parenchyma contain- 
ing starch within its chloro- 
plasts; e, vascular bundle; d, 
d, stomata. 



152 



CONSTRUCTION OF PLANT S FOOD 



photosynthesis varies with the light intensity up to and 
even beyond full sunlight. Nevertheless a very feeble light 
is sufficient to sustain photosynthesis to a slight extent, and 
it may be that in nature plants get as much usable en- 
ergy for this function out of the diffuse light from all quar- 
ters of the sky as from the direct sunlight itself. In dwellings 
where plants stand before a window they are lighted by only 
a small part of the sky, and this rapidly 
diminishes the farther back plants are 
placed, so that only a few feet from a 
window they may be actually losing in 
weight for lack of sufficient food con- 
struction, although they may be grow- 
ing and having the appearance of some 
thrift. 

Time Required. — Engelmann's bac- 
terium method shows that photosyn- 
thesis begins instantly on exposure to 
light. It is found, however, that it 
takes several minutes, and in some cases 
an hour or more before starch appears 
in the chloroplasts ; and this is evidence 
that this starch represents carbohy- 
drate that is formed faster than it is 
being carried away — a surplus that 
would hinder the constructive process if allowed to remain in 
solution in the cell sap. And the starch in the chloroplasts 
might be of direct benefit in increasing the chloroplastic sur- 
face and in refracting and reflecting the light so that more of 
it would be retained within the body of the chloroplast. 

Carbon Dioxide. — It is found that the very small percentage 
of carbon dioxide in the atmosphere is not the optimal amount 
for photosynthesis ; for this function rises in activity as the 
CO 2 content is increased from .03 per cent, to 8 per cent., and 
with this increase photosynthesis can go on even with closed 
stomata. About one half of the dry weight of a plant is car- 




Fig. 81. Cross-section of 
a portion of leaf of Codo- 
nanthe, showing the water- 
storage tissue at f, and the 
chlorophyll-bearing tissues at 
e. (After Schimper.) 



PHOTOSYNTHESIS IN LOWER PLANTS 153 

bon, and it is of great importance that this so necessary ele- 
ment in nutrition is in an extremely mobile condition and 
capable of distribution by the unceasing currents of the atmos- 
phere. 

Water. — The photosynthetic cells are like all others in being 
able to perform their functions better when in a state of tur- 
gidity. It is found, however, that isolated cells are able to 
photosynthesize to a certain extent when in a flabby or even 
plasmolyzed condition ; and mosses and lichens are remarkable 
for their power of photosynthesis even after they have begun 
to dry up. But the higher plants, with their waterproof epi- 
dermis, are unable to photosynthesize after the reduction in 
the water content has caused the closure of the stomata, and 
thus prevented the continued inflow of carbon dioxide. 

Temperature. — The temperature may rise too high or fall 
too low for photosynthesis ; but, as might be anticipated, plants 
in different latitudes are not affected alike by the same tem- 
peratures. For instance, in the tropics photosynthesis ceases 
when the temperature becomes as low as 8° to 4 C, while 
in the subtropics and temperate zones to produce a like result 
the temperature must fall almost or quite to o° C. ; and cool- 
temperate, arctic and alpine plants continue photosynthesizing 
until they become frozen. In regard to the maximum tem- 
perature, plants in general cease photosynthesizing after long 
exposure to approximately 38 ° C. 

Photosynthesis in the Lower Plants. — In the simpler 
Algse where each individual consists of a single cell, a chain 
of cells forming a filament, or a thin lamina one cell thick, 
each cell contains one or more chloroplasts and carries on food 
synthesis. In Pleurococcus the chloroplasts are large in com- 
parison with the size of the cell and seem nearly to fill the cell 
cavity. In CEdogonium and Nitella the chloroplasts are 
numerous and relatively small. In Spirogyra each cell has 
one to few chloroplasts each in the form of a spiral band (Fig. 
82). In these as well as in the higher plants the chloroplasts 
lie embedded in the cytoplasmic layer surrounding the vacuole 
and lining the cell wall. 



154 



CONSTRUCTION OF PLANT'S FOOD 



In liverworts and mosses the photosynthetic tissue reaches 
a fair degree of differentiation. The thallus of Marchantia 
polymorpha has beneath its upper epidermis groups of chloro- 

plast-bearing cells that cor- 
respond to the palisade cells 
in the leaves of higher plants 
(Fig. 83). Each of these 
groups is contained in a 
diamond-shaped compart- 
ment as seen from the sur- 
face, the partitions at / being 
a single cell in breadth, and 
each compartment commu- 
nicates with the outer air 
through an unusually large 
stoma. Each group of pho- 
tosynthesizing cells is there- 
fore in a separate air cham- 
b e r . The photosynthetic 
product is delivered to color- 
less parenchyma cells in the 
lower half of the thallus. 
In Marchantia the thallus 
lies flat upon the ground, 
and the photosynthesizing cells and stomata can function to 
best advantage where we find them in the upper part of the leaf. 




Fig. 82. Chloroplasts from different 
sources. A, Pleurococcus, with chloroplast 
at c; B, cell of Spirogyra, with spiral 
chloroplast at c, nucleus at n, and pyrenoid 
at e. C, cross-section of Spirogyra cell, 
with nucleus at n, and section of chloro- 
plast and pyrenoid below. D, cell of 
CEdogonium, with numerous chloroplasts. 
All highly magnified, but not to the same 
scale. 




Fig. 83. Cross-section through the thallus of Marchantia. j, stoma leading into 
a relatively large air-chamber in which arise numerous cells with chloroplasts, k; I, I, 
cells forming partitions between the air-chambers; m, cells destitute of chloroplasts 
and with numerous oblong pits; h, upper, and i, lower epidermis. (After Sachs.) 



PHOTOSYNTHESIS IN LOWER PLANTS 



155 



In Sphagnum the leaf is one cell thick (Fig. 84), and the 
photosvnthetic cells are arranged in the form of an open mesh- 
work as seen from the surface and between them are large, 
clear, water-storage cells hav- 
ing minute pores through the 
outer wall at the under sur- 
face through which water can 
be imbibed. As seen in the 
figure the photosvnthetic cells 
are elongated parallel with the 
length of the leaf and are 
thus adapted to conduct away 
their own products. 

In Polytrichum there are 
vertical chains of photosvn- 
thetic cells, but there is no 
closed upper epidermis, as 
shown in Fig. 85, and these 
cells are freely exposed to the 
outer air excepting when the leaf, in danger of losing too 
much water, rolls up from both edges, so that the free margins 
of the incomplete epidermis touch or overlap. Here as in 




54. Portion of leaf of Sphag- 
num, in cross-section on the left, and 
surface view on the right. h, hole 
through the wall; i, chlorophyll-bearing 
cells; /, water-storage and water-con- 
ducting cells, with annular thickenings 
at ft. (After Strasburger.) 




Fig. 



Cross-section through a portion of leaf of Polytrichum commune, b, chains 
of chlorophyll-bearing cells; d, bast fibers. (After Strasburger.) 



Marchantia the products of photosynthesis are given over to 
elongated conducting cells in the lower part of the leaf. 

In many mosses the blade of the leaf on either side of the 
midrib is one cell thick and all of these cells contain chloro- 



I 5 6 CONSTRUCTION OF PLANT'S FOOD 

plasts, and being elongated parallel with the long axis of the 
leaf they conduct away their own products (Fig. 86). 

Synthesis of Food Without Light. — Some kinds of bac- 
teria (Nitrococcus and Nitrosomonas) inhabiting the soil oxi- 
dize ammonia and its compounds to nitrous acid and its salts 
and utilize the kinetic energy liberated by this oxidation in 
making food from carbon dioxide and water. Other bacteria 
included in the genus Nitrobacter oxidize the nitrous to nitric 
compounds and get energy in this way for food synthesis. 
Still other bacteria utilize the energy from the oxidation of 
salts of sulphur and iron occurring in the soil. 

In green plants the construction of nitrogenous foods by 
uniting carbohydrate made in the leaves with salts of nitro- 
gen, and sometimes in addition, with salts of sulphur and phos- 
phorus brought up from the soil, can take place in darkness as 
well as in the light, and possibly in any living cell. When 
proteid synthesis goes on in darkness the energy for the work 
apparently comes from the decomposition of a part of the car- 
bohydrates, in which event sunlight would still be, although 
indirectly, the source of the energy. 

While the synthesis of nitrogenous foods presumably can 
take place in any living cell there are reasons for believing that 
most of this work is done in the leaf. Mature leaves at the 
height of their activity contain large amounts of the amide 
asparagin that hardly can be accounted for except by the theory 
that they are manufactured there. Although there is more 
asparagin present in leaves in the evening than in the morning 
this does not necessarily imply that light is required for its 
production, for both asparagin and carbohydrates would dif- 
fuse out of the leaf during the night, and the production of 
asparagin could not be kept up for lack of carbohydrates which 
are needed in its manufacture, and which we know can only 
be made in the light. 

Although approximately four fifths of the atmosphere is 
nitrogen the vast majority of plants are unable to use it in its 
uncombined form for food construction, and for this purpose 



CONDITIONS AFFECTING PHOTOSYNTHESIS 



157 



it must be taken, in the case of green plants, mostly in the form 
of some nitrate, such as calcium or potassium nitrate. The 
case is different with saprophytic plants, such as the toadstools 
and their kind, moulds, and many forms of bacteria, for these 
plants can appropriate for food various nitrogen compounds 
in the excreta and dead bodies of other plants and animals. 
Parasitic plants, such as the 



rusts, mildews, 



blights 



and 




Fig. 86. CroSs-section, A, and surface 
view, B, of a leaf of common moss, showing 
chloroplasts, c. 



smuts, and those of higher 
order, such as Cuscuta, ap- 
propriate the food of the 
plants upon which they are 
parasitic. 

Although the green plants 
and plants in general are un- 
able to appropriate the free 
nitrogen of the air, there are 
a few forms of bacteria 
which have this power, such 
as Clostridium Pasteurianum 
living in the soil, and tubercle 
bacilli that cause and inhabit the tubercles on the roots of Legu- 
minosse and some other families of plants. These occur nor- 
mally in the soil in most localities, and enter the roots through 
the root hairs and furnish the stimulus for the growth of the 
tubercles. While these bacteria are parasitic on the green plant 
to the extent of utilizing carbohydrate made by it, they never- 
theless are useful to the green plant in that they compound the 
free nitrogen of the air into substances which the green plant 
can use for food. For this reason the soil in which leguminous 
plants are grown, such as peas, clover, alfalfa, etc., becomes 
richer in nitrogen, even when the crop is harvested and only 
the stubble and roots are left to decay and become a part of 
the soil. 

Restating briefly the relation of carbohydrate to nitrogen- 
ous foods : carbohydrates, such as sugars and starches, are 
formed in the chloroplasts by the use of the sun's energy 



15^ CONSTRUCTION OF PLANT'S FOOD 

directly. Later some of these carbohydrates are united with 
salts of nitrogen to form nitrogenous food substances, and 
from these still more complex nitrogen compounds are formed 
with the addition of elements from the salts of sulphur and 
phosphorus. While the energy for the production of carbohy- 
drates must be taken directly from the sun (with the exception 
of the few bacteria that produce nitrous and nitric compounds 
from ammonia and its salts, and those that oxidize salts of 
iron and sulphur as above stated), the energy for the com- 
pounding of nitrogenous foods is taken indirectly from the sun 
(and hence can go on in darkness and in saprophytes and para- 
sites), by the decomposition through oxidation, or otherwise, 
of carbohydrates and substances derived from them. 

Illustrative Studies 

1. Study chloroplasts in a moss leaf. Mount a fresh leaf in 
water and study it with low and high powers. Note the posi- 
tion of the chloroplasts within a cell and count them. Put a 
drop of chloral hydrate-iodine (see under this title in Chapter 
XV) on the slide against the coverglass and as the reagent 
diffuses under watch its progressive reaction on the chloro- 
plasts. Is starch demonstrated ? In precisely what part of the 
cell did it occur? Measure one of the starch grains. Draw 
a single cell as seen before and after application of chloral 
hydrate. 

2. Study Spirogyra in the same manner and draw a single 
cell as before. 

3. Make free-hand cross-sections of some leaf and mount 
the thinnest — even the smallest fragments — in a drop of water. 
Find the palisade and the spongy parenchyma. Do the chloro- 
plasts have the same position in the cells as those of the moss 
leaf were found to have? Treat with chloral hydrate-iodine 
as above and watch for the first indications of starch. Do 
you find evidence that the starch was formed by the chloro- 
plasts? How large are these bits of food that have just been 
compounded from C0 2 and water? Draw a portion of the 
section from one surface to the other as seen before and after 
the chloral hydrate reaction. 



ILLUSTRATIVE STUDIES 159 

4. Study in the same way leaves from plants that have been 
kept in the dark for forty-eight hours, and leaves from plants 
that have been kept all day in strong diffuse light but in an 
atmosphere from which C0 2 has been absorbed. To do this 
set a potted plant on a glass plate; put a stick of KOH in a 
bottle beside it; place a tubulated bell-jar over all and seal it 
with sealing wax to the glass plate; stop the opening in the 
bell-jar with a perforated cork, and insert through the cork a 
glass tube bent at right angles, and into the horizontal arm 
of this place loosely small lumps of soda lime. Now air can 
enter but the C0 2 will be absorbed from it by the soda lime, 
and that C0 2 evolved by the respiration of the plant will be 
absorbed by the KOH under the bell-jar. 

Arrange a check experiment in all respects like this one 
except that sawdust is to take the place of the soda lime and 
the KOH under the bell- jar is to be omitted. It will not do 
to place these plants in direct sunlight because the air in the 
bell- jar would become too hot. The plants used in this ex- 
periment must have been kept in the dark for forty-eight 
hours to eliminate starch from the leaves. Before the experi- 
ment and at its close test sections of leaves from both plants 
with chloral hydrate-iodine. 

5. With a brush coat half of the under side of some leaves 
having stomata on the under side only, with melted beeswax 
one part and cocoa butter one part, after the leaves have been 
covered from the light for forty-eight hours. The leaves are 
to remain on the plant through this experiment. Scratch 
through the upper epidermis with a needle in a few places on 
the upper side of the coated half of some of the leaves. Now 
expose the leaves to the light for half a day and then pick 
them off and plunge them into cold water and peel off the 
wax. Extract the chlorophyll from the leaves in boiling 
alcohol and place them in a solution of iodine. Do the two 
halves show starch alike? What do you note along the 
scratches? What do these observations teach? Make col- 
ored drawings to show your results. 



CHAPTER X 

CIRCULATION OF FOODS THROUGHOUT THE PLANT 

Need of Circulatory Tissues. — As we have seen in Chap- 
ter IX, most of the food is manufactured in the leaves and 
must be carried from them throughout the plant wherever it is 
needed for supplying materials and energy for growth and 
repair or other purposes, or where food is to be stored up for 
future use. The higher plants, namely, the Vascular Crypto- 
gams, Gymnosperms and Angiosperms, have attained to such 
size that the distance to be traveled by the food is often great ; 
and it has been found that short cells such as occur in the meri- 
stematic cells of growing points, or in the pith or outer bark 
of older parts will not suffice for carrying food except through 
very short distances. The sieve tubes and associated cells of 
the phloem alone are able to do this. Without them the con- 
duction of food could not take place any more than water could 
be carried in sufficient amounts without tracheal tubes and 
tracheids. 

In following the phylogeny of tissues in the lower plants 
we find that the evolution of the food-conducting, as well as 
water-conducting, tissues is clearly in correlation with the evo- 
lution of leaves, which by their efficiency in food-making, and 
their large transpiration surfaces, have created a demand for 
means of conducting both food and water more rapidly than 
can be done by short unperforated parenchyma cells. In the 
mosses, represented by Polytrichum commune, for example, 
these conducting tissues have made a distinct beginning. Here 
the center of the stem is occupied by a vascular bundle of the 
concentric type (see page 44) with the water-conducting sur- 
rounded by the food-conducting tissues. 

The development of the phloem from the procambium has 

160 



CIRCULATION OF FOODS l6l 

been told in Chapter 1 1 ; but it will be useful here to review the 
different elements of the phloem and give what is demon- 
strated or conjectured on good grounds to be their functions. 

The sieve tubes ( Fig. 18), it will be remembered, are formed 
by the perforation of the end walls in vertical rows of cells, 
so that the row becomes essentially a tube through which all 
kinds ni foods can flow in solution with less interruption than 
through relatively short cells with walls intact. The sieve 
tubes are especially well adapted for the conduction of pro- 
teids. which are colloidal and diffuse throughout the cell cavity 
and through division walls with difficulty. It can be shown 
that the contents of the sieve tubes can flow en masse through 
the perforated partitions from one member of the tube to an- 
other, for when a stem is cut off the contents of the sieve tubes 
flow out until the latter are at least partially emptied for one 
or more internodes back from the cut, and in flowing this dis- 
tance the contents must in some instances have passed through 
hundreds of partition walls. 

The companion cells and sieve tubes are formed by the lon- 
gitudinal division of a common mother cell, and they continue 
in intimate association. When the walls of the companion 
cells become appreciably thickened they are frequently pitted 
where they are in contact with the sieve tubes or surrounding 
parenchyma or medullary ray cells. The companion cells are 
therefore adapted to take over materials from the sieve tubes 
and deliver them to tissues that can carry them where they are 
wanted for immediate use, or where they can be stored for con- 
sumption later on. What part the companion cells play in the 
longitudinal transmission of foods has not been worked out. 

The cells of the phloem parenchyma appear to take an active 
part in the longitudinal transmission of carbohydrates and 
amido-acids, and through their pitted communications with 
the medullary rays they send foods of all kinds into the rays 
for radial distribution and storage; and they themselves, to- 
gether with the phloem parts of the medullary rays, are the 



1 62 CIRCULATION OF FOODS 

chief place of storage of proteids during- resting periods of 
winter or dry seasons. 

As was stated in Chapter II, all classes of plants do not pos- 
sess the full complement of phloem elements here described. 
In Gymnosperms and Vascular Cryptogams the companion 
cells do not occur, and their place is taken by vertical rows of 
parenchyma cells ; and in Monocotyledons the parenchyma cells 
are lacking. Neither are the sieve tubes alike in all respects 
in the different classes and families of plants. In the Gymno- 
sperms the primary end walls of the sieve tube members (cells 
composing the tubes) are not dissolved away at the bottom of 
the pits, as seems to be the rule in Angiosperms, and in the 
latter class the size of the pores varies greatly in plants of 
different habits of growth. 

For the study of sieve tubes the squash and its allies, or the 
grape, hop, or other climbing plant is chosen, because, in these 
plants with slender stems, the sieve tubes and the pores through 
the partition walls are found to be larger than in plants of 
different habit, evidently for the reason that, the stem being 
slender and the crown of leaves relatively large, the tissues 
devoted to food-transportation must be unusually efficient. In 
many plants the pores in the sieve plates or partition walls are 
discerned with the greatest difficulty because of their minute- 
ness, and in some cases they can not be made out at all. 

Evidence that the Phloem Carries the Food. — Micro- 
chemical examination, in which chemical reagents are applied 
to sections under the microscope, shows that the phloem is 
filled with food substances. Chemical analysis of the ex- 
pressed contents of sieve tubes has found 7 to 10 per cent, of 
solid or dissolved substances, of which 20 per cent, was pro- 
teid, 30 per cent, amide (a nitrogenous food of simpler com- 
position than proteid), and 38 per cent, soluble carbohydrate. 
That these substances are actually transported by the phloem 
is shown by experiments in which the continuity of the phloem 
is wholly or partially interrupted. When a ring of bark is 
removed down to the phloem there is no significant disturbance 



TRACHEAL TISSUES ASSIST PHLOEM 1 63 

in the circulation, as shown by the fact that growth in diame- 
ter continues below the girdle as well as above, and the storage 
of food goes on in storage cells on both sides of the girdle alike. 
When, however, the ring of bark is removed clear to the wood, 
so that the possibility of longitudinal flow through the phloem 
is prevented, it is- found that growth ceases or is greatly less- 
ened below the girdle and the storage of food below the girdle 
is prevented. Again, when a willow branch, for example, is 
cut off, girdled close to its lower end, and placed in a vessel of 
water, adventitious roots spring out much more abundantly and 
vigorously above the girdle than below it ; and when the girdle 
is incomplete, so that it is spanned by a strip of phloem, roots 
spring out in line with the strip above and below the girdle 
alike. When girdling is performed on plants with bicollateral 
bundles (see page 44), as in the Cucurbitacese, the phloem 
parts next to the pith are, of course, left intact and the food 
is found to travel up and down without hindrance. The evi- 
dence is therefore conclusive that the phloem is the highway 
for the vertical transmission of food through the stem. 

In those Monocotyledons where the vascular bundles are 
scattered promiscuously throughout the stem the phloem can 
not, of course, be removed by girdling, since every bundle, 
near the periphery or remote, consists of both phloem and 
xylem. 

Evidence that the Tracheal Tissues Assist the Phloem 
in the Upward Transmission of Food. — No tissues of the 
plant body are so well adapted for the rapid transportation of 
food upwards as the tracheal tissues, consisting of tracheal 
tubes and tracheids, for in them are currents of water moving 
much more rapidly than is possible in the phloem. The evi- 
dence that these tissues are so used is found in the chemical 
analysis of their contents and in the results of girdling. 
Chemical analysis shows for trees that early in the spring, 
when a wound results in bleeding, water from the tracheal 
tissues contains in solution sugar, proteids, and amides. At 
other times of the year these may be present there but in less 



164 



CIRCULATION OF FOODS 



'• 



amount. The maples and birches are notable examples of 
this, while the grape is an exception, for the sap gathered 
from the bleeding of grapevines, after their pruning in early 
spring, contains solutes from the soil, but not stored food- 
stuffs, as in the case of maples, birches, box elders, etc. 

The evidence afforded by girdling that 
the tracheal tubes carry foods upwards in 
the spring is this : If girdling is done 
on trees in the fall, winter, or spring, 
before the resumption of growth sets in, 
and while wood and bark are still stored 
full or reserve food, nevertheless the 
storage cells below as well as above the 
girdle are emptied of their supplies after 
the spring growth begins, although the 
only tissues left capable of carrying the 
reserve foods past the girdle are the 
tracheal tissues in the wood (Fig. 87). 
Furthermore, when the main stem of 



Fig. 87. Diagram 
to show path of stored 

food upward through an inflorescence is girdled near its base 

the tracheal tubes, and 

through the phloem and the exposed wood is prevented from 

portion of the bark, d • b d f jj & H w ^J 

and showing how this J o j o J 

passage is not pre- the fruit goes on to maturity. In this 
case also the tracheal tissues afford the 



vented by girdling. 



only possible channels for carrying past the girdle the large 
amounts of food needed for the growth of the fruit and for 
storage in the seeds. 

The inference does not follow from these observations that 
the phloem is not needed and is not employed in the upward 
transportation of foods. The only thing proven by the gird- 
ling experiments is that when the continuity of the phloem 
is thus broken the tracheal tissues can carry food upwards in 
sufficient quantities past the girdle. There are, on the other 
hand, anatomical facts to show that the phloem is employed in 
the upward movement of foods. In inflorescences, for in- 



RELATION OF PHLOEM ELEMENTS 



I6 5 



stance, which make no food but use large amounts that must 
be brought upwards into them, the phloem, and particularly the 
sieve tubes, attain to a greater relative development than in 
any other parts of the plant. 

Relation of Phloem Elements to Other Tissues. — The 
iond in its downward or upward course through the phloem 
may be drawn upon at any point by living tissues for growth, 
repair, etc., or it may be set 
aside for storage in medullary 
rays, xylem and phloem par- 
enchyma. Evidently to facili- 
tate this movement of food- 
stuffs there are often pores 
between sieve tubes and com- 
panion cells, and thin places 
or pits where the medullary 
rays abut on companion cells 
or phloem parenchyma. Pits 
also occur in the tangential 
walls of the medullary rays 
to help along the radial move- 
ments and storage of foods 
in the medullary rays; and 
pits in the walls separating 
the rays from wood paren- 
chyma cells assist in the trans- 
mission of foods to, and stor- 
age in, the latter (Fig. 88). 

The medullary rays have for their primary function the 
radial transmission and storage of food. Their intimate rela- 
tion with the cells of the phloem at their outer end and with 
the xylem parenchyma along their inner course, and the fact 
that we usually find them gorged with food, points to this con- 
clusion. The short vertical extent of the rays, and their iso- 
lation from each other, renders them unsuited for the vertical 




Fig. 88. Showing pitted connections 
between medullary rays and xylem paren- 
chyma, and between contiguous xylem 
parenchyma cells. m, medullar}' rays; 
11, xylem parenchyma. Camera-lucida 
drawing of tangential section of wood 
of yellow poplar. 



1 66 CIRCULATION OF FOODS 

or longitudinal transmission of foods. If they were of value 
in this respect girdling would not prevent the downward flow 
of foods. 

The extreme frequency of the rays is one factor of great 
importance to their efficiency in radial conduction and storage. 
In tangential section, that is, as one would see it when facing 
a tree, there are approximately between 20 and 30 medullary 
rays in every square millimeter (Fig. 
89) ; so that when the leaves are at the 
height of their food-construction it may 
be inferred the rays are very active in 
relieving the sieve tubes of their loads. 

The Course of Food Distribution. — 
The logical place to begin the discussion 
fig. 89. Low-power, of food distribution is where the food 

LTgTntt^^ectTon 116 of firSt C ° meS {nt ° beil1 ^ in tHe P alisade and 

wood of oak, to show spongy parenchyma of the leaves. It will 
^requency^o^me^u^ary ^ remembered (see page 117) that the 

mm. square. The num- veins ram jf y throughout the leaf SO ex- 

ber of rays shown is •* ° 

below the average for tensively that the last branches probably 

woody plants. .-, „ , ^ 

average no more than .2 mm. apart; so 
that food manufactured in cells farthest from these branches, 
namely, those half way between them, would need to travel 
laterally about .1 mm. before entering the border parenchyma 
cells of the veins in which it begins its journey out of the leaf 
(Fig. 58, E). These border parenchyma cells have already 
been told about on page 45, and their relation to the other food- 
conducting cells of the veins will now be given more in 
detail. 

The vascular bundles in the larger veins of the leaf may 
have all of the elements in their phloem part that occur in the 
stem from which they spring; but as the veins in branching 
get smaller and smaller the phloem parenchyma is left behind, 
while the sieve tubes and companion cells remain ; then farther 
along these do not appear and their place is taken by elongated 



COURSE OF FOOD DISTRIBUTION 1 67 

cells that are apparently the undivided mother cells of sieve 
tubes and companion cells (see page 38) ; then these are left 
out, and the ends of the veins have only border parenchyma 
cells (which are morphologically a part of the mesophyll or 
fundamental parenchyma and not of the vascular bundles), 
surrounding the last tracheids. These facts are represented 
diagrammatically in Fig. 90. 

The food from the palisade and spongy parenchyma in the 
form of soluble carbohydrate such as grape sugar, or in sol- 



W^rr rt p=p= h . 



nr irp i 



■ , . ' . ■■ <- 



Fig. 90. Diagram indicating the succession of the conducting tissues of a vein 
from the base towards the apex, a, border parenchyma; b, companion cells; c, sieve 
tubes; d, undivided mother-cells of companion cells and sieve tubes; e, tracheal elements. 



uble nitrogenous form such as asparagin and other amides, 
and even in the more complex form of soluble proteids, passes 
by diffusion into the border parenchyma cells of the smallest 
veinlets, and then begins its movement, still by diffusion and 
other ways about which we have no exact knowledge, towards 
the larger veins which are to carry it out of the leaf or deliver 
it to the midrib for transportation. A very simple demonstra- 
tion shows that the food takes this course. After a day of 
active photosynthesis the leaves are loaded with starch at sun- 
down. A leaf removed at this time, bleached in boiling alco- 
hol, and stained with iodine, shows the blue starch reaction 
all over. By sunrise the next morning, however, a leaf when 
removed and treated in the same manner takes on a yellowish 
color, showing that the starch has disappeared. But if one 
of the principal veins is cut in the evening that portion of the 
leaf between the cut and the extremity of the vein, which of 
course would be the part tributary to this vein, still retains 
its starch in the morning, while the rest of the leaf where the 



1 68 



CIRCULATION OF FOODS 



veins are left intact have been emptied (Fig. 91) ; and when all 
of the principal veins are cut through at their base the entire 
leaf is filled with starch in the morning. Clearly the cutting 
of the veins broke the continuity of the highways through 
which the food passes out of the leaf. 




Fig. 91. Showing the effect of cutting across the veins on the removal of food 
from the leaf. A, all of the main veins are cut across near their bases; B, the 
mid-vein alone has been severed. The stippled areas indicate the starch reaction with 
the iodine test. (After Mary Blue.) 



COTRSF OF R)()l) DISTRIBUTION 



69 



The passage of the food through the border parenchyma 
cells of the smallest veinlets by diffusion must be very slow, 
but since there is a multitude of these veinlets (approximately 
6,000 to the square centimeter) tributary to the larger veins 




upward 



Tracheal tube carrying food upward 
from the leaves 



Fig. 92. Diagram illustrating the descent of food from the leaf into the stem, 
and its circulation upwards and downwards through the sieve tubes, and upwards 
through the tracheal tissues. 



where the sieve tubes occur, it is plain that a slow movement 
in the many veinlets could feed a much more rapid flow in the 
few main channels ; and this is presumably what happens. 

When the food reaches the branch which bears the leaf it 
may pass through the phloem down or up, or part may pass 
down and part up at any given moment. Some of the food 
may also be transferred to the water tubes in which it will be 
hurried along to the growing apex of the branch or to fruits 
and seeds in course of formation, and be put to immediate 
use (Fig. 92). When the branches are still growing in length, 
or when fruit is developing, a great deal of the food goes up 
to nourish the new growth. After growth in length has 
ceased, and if fruit is not forming, doubtless most of the food 
goes downward to sustain the cambium in its production of 
new tissues, or for storage until demand is made for it at 
the time of flowering and fruiting in annuals, or when growth 
is resumed in the spring in the case of perennials and biennials. 



i;o 



CIRCULATION OF FOODS 



In Indian corn, for instance, after blossoming- the food is 
for the most part sent into the ears, and that which is made 
by the lower leaves passes up, and that by the upper leaves 

down, to the ears for 
storage in the form of 
starch, proteids, and 
oils (Fig. 93). 

In perennial plants, 
such as trees, part of 
the food made in spring 
and early summer is 
used at once in growth 
in length and diameter 
of branches, trunk and 
roots ; but by the begin- 
ning of summer, or 
even in May, much of 
the food is being stored 
in roots and trunk. 

By August growth 
has almost ceased in 
most woody plants, and 
the bulk of the food 
made thereafter is 
stored in branches, 
trunk, and roots for 
use during the winter 
to a certain extent, and 
for resumption of 
growth in the spring. In accordance with this the flow of 
the food would be up as well as down in the first part of the 
period between the appearance and fall of leaves, and chiefly 
down for storage after the elongation of the branches and the 
growth of fruit has practically ceased. 

Most of the reserve food is stored in the roots and trunk, 
and in the spring the larger part must ascend where growth 




Fig. 93. Diagram showing how, in Indian 
corn, the food from the upper and lower leaves 
finds its way into the ears. 



COURSE OF FOOD DISTRIBUTION 1 7 I 

and fruitage is going on in the crown, and where cambial 
activity is first awakened. The sieve tubes which are empty 
during the winter can be rilled by these ascending currents, 
and in most instances the tracheal tubes are also pressed into 
service and carbohydrates, chiefly as sugar, and proteids and 
amides to a certain extent, are poured into them from their 
place of storage in the xylem and phloem parenchyma and 
medullary rays. The tracheal tubes must prove very efficient 
carriers, for the ascending currents of water would sweep the 
food along much faster than it could be moved in the sieve 
tubes. While most plants make large use of the tracheal tis- 
sues in this way there are others, such as the grape, which 
make little or no use of them and send practically all of their 
food into the sieve tubes for transportation. The fears that 
the grapevine may be depleted of its food when bleeding from 
spring pruning are therefore groundless, since little more than 
water and a small percentage of salts from the soil are then 
lost. 

It will be noted that just as the sieve tubes can carry food 
up or down as needed, so the medullary rays are able to trans- 
port foods radially inward for nourishing the xylem or for 
storage, and outward again when they are to be distributed 
and put to use. There is nothing about the construction of 
the food-conducting tissues to prevent movement in them in a 
certain direction at one time and in the opposite direction at 
another, and it is quite possible that a flow in opposite direc- 
tions may occur at the same time in a cell or tube. 

The growth of seedlings in their earlier stages presents 
another set of conditions in food distribution. Then the food 
must move from its place of storage in cotyledons and endo- 
sperm before the special food-conducting tissues have become 
differentiated; but the distance to be traveled is short and the 
cell walls are new and thin and offer relatively little resistance. 
Under these conditions it seems that food can travel fast 
enough to maintain rapid growth without the aid of sieve 
tubes. In seedlings the direction of flow of food is down 



I7 2 CIRCULATION OF FOODS 

into the roots and up into the shoot from the place of storage, 
but as soon as the green leaves unfold they at once become an 
additional source of food supply, and by that time the vas- 
cular bundles have been formed and the sieve tubes are ready 
to take up their work, carrying the food down or up as need 
compels. 

The anastomoses of the vascular bundles described on page 
43 are of great use in the distribution of food as well as of 
water. In the elm tree, for instance, the leaves are two 
ranked, so that two sides of the branch are throughout its 
length bare of leaves ; and the smaller branches arise from the 
larger also in two rows, so that if the food descending from 
the leaves took a straight course down the branch this would 
be without nourishment throughout more than half of its body 
since the food travels with difficulty out of one bundle through 
intervening tissues into another as shown on page 183. But 
this difficulty does not exist in uninjured plants because the 
bundles are so knit together by anastomoses (Figs. 20 and 
49, B), that the food from one side of a stem can be shunted 
to another side whenever there is need. Likewise in Indian 
corn, as a rule more corn is produced on one side of the stalk 
than on the other, but the more fruitful side has taken contri- 
butions from the leaves of the opposite side through the 
numerous anastomoses at the nodes. 

These examples will serve to illustrate the general statement 
that whenever, or for whatever cause, one side of a plant 
requires food that the leaves on that side are unable to furnish 
all other sides may be drawn upon, since all the vascular bun- 
dles are knit together in one system. 

The course of food distribution will now be summed up : 
When growth begins in the spring food is carried to the un- 
folding buds upward through the sieve tubes and tracheal 
tissues from its place of storage in branches, trunk, and roots. 
Soon the leaves have grown forth and begin the construction 
of new food. This flows out of the leaf through the border 
parenchyma cells and phloem, and on reaching the branch it 



ANNUAL ADDITIONS TO TISSUES 



173 



takes the direction compelled by the plant's need. It may flow 
down through the phloem or up through the phloem and 
tracheal tissues. 

Whether the food flowing down or up shall he used at once 
or stored For future use depends on the need of the plant. So 
long as growth is rapid, and during the period of flowering 
and fruiting- large quantities of food are put to immediate use. 

As the food travels down or up through the phloem a part 
of it is removed from these longitudinal highways for the 



Tracheal tube carrying food upward 
Xylem parenchyma receiving 

food from medullary rays 
and storing it 



Sieve tube carrying food 
downwards from 
the leaves 




Medullary ray cells carrying food inward and outward 
from the sieve tubes The ray cells also store food 



Parenchyma 
storing foe* 



Fig. 9}. Diagram showing the circulation of food through the sieve tubes, medul- 
lary rays and tracheal tubes, and its storage in the parenchyma cells of the wood and 
bark. The black bodies in the cells indicate stored food. 



immediate nutrition of the cambium and part is carried farther 
inward by the medullary rays and used by them and other 
living cells of the wood in nutrition, or some is stored in the 
rays and wood parenchyma and cells of the pith immediately 
bordering the wood (Fig. 94). Some of the food taken from 
the phloem is distributed outward to the living cells of the 
cortex and pericycle (see page 32), and used in nutrition or 
temporarily stored. In short, all living cells of the plant body 
draw upon the supplies that are in circulation throughout the 
phloem. 

Annual Additions to the Food-Conducting Tissues. — In 
the spring when growth in perennial stems and roots is re- 
sumed the cambium devotes itself first to the production of 
water-carrying tissues, as related in Chapter VI. But at the 
same time, although to a much less extent, it begins to lay 



174 CIRCULATION OF FOODS 

down new phloem elements. Early spring additions to the 
phloem seem to be more imperative in some plants than in 
others, because in some the sieve tubes are functional but a 
single year and the advent of their second spring finds them 
empty and their sieve plates blocked by an accumulation of a 
peculiar, highly refractive substance called callus, and in this 
condition they remain until crushed out of existence by the 
growth of surrounding tissues. In other plants, such as the 
grape, although in the spring the old sieve tubes are empty 
and dammed up by callus the latter is soon dissolved away 
and the tubes again are filled from stored materials in phloem 
parenchyma and medullary rays. 

The cambium ceases its additions to the xylem or wood 
side of the bundles early in August, but it may continue to 
slow additions to the phloem until the close of the growing 
season. 

As to the length of life of the phloem elements, in some 
plants the sieve tubes live but a single year while in others 
they may survive and remain functional for a few years at 
most. The companion and phloem parenchyma cells may die 
with the sieve tubes, but in some cases they survive these, even 
until they become cut off from the general circulation during 
the formation of borke (see page 57). Therefore in stems 
several years old we are apt to find the outer and older por- 
tions of the phloem collapsed and dead (Fig. 24). 

Relation of One Year's Phloem Elements to those of 
the Next. — Fig. 95 shows diagrammatically how the phloem 
of one year narrows down at the close of the year's growth 
in length and lies in immediate contact with the primary 
(earliest-formed) phloem of the following year. The ending 
of the year's phloem (where it tapers to a point in the dia- 
gram) consists of sieve tubes and companion cells in Dicoty- 
ledons, and sieve tubes and vertical rows of phloem paren- 
chyma cells in Gymnosperms; and these join with the corre- 
sponding elements that are first differentiated from the pro- 
cambium of the succeeding year's growth in length. It will 



CHARACTER OF CIRCULATING FOOD 



175 



be seen by this diagram that the food highways in the leaves 
have direct communication with the primary phloem in the 
now segment of stem which bears 
them, and this primary phloem is 
in turn continuous with the phloem 
elements which the cambium builds 
the current season throughout 
branch, trunk and roots. 

As has been said, in some plants 
the sieve tubes are functional for 
one year only ; in others for two or 
more years, and it would therefore 
depend upon the kind of plant to 
what extent the phloem strands in 
the older annual segments (2, 3, 
of the diagram) assist in carrying 
food the current season. It is pre- 
cisely because the youngest and 
younger phloem tissues are the 
most active that, in girdling, the 
bark can be stripped off nearly to 
the wood without apparently hin- 
dering the flow of food. 

Character of the Circulating 
Food. — The nitrogenous foods 
circulating in the sieve tubes and 
other parts of the phloem are 
mostly in the form of asparagin 
and other amides which are soluble 
and more diffusible than the solu- 
ble proteids. The insoluble pro- 
teids must be changed to the solu- 
ble condition before they are capa- 
ble of translocation. 

The carbohydrates are mostly 
in the form of glucose (grape 




Xylem 

Fig. 95. Diagram to show the 
relation of the food-conducting 
tissues of the leaf to those of 
the stem; and in the stem the 
relation of these tissues of one 
year to those of preceding years. 
The dotted line between phloem 
and xylem stands for the cam- 
bium. The figures at the bottom 
of the diagram indicate the age 
in years of the zones of tissues in 
phloem and xylem. 



176 CIRCULATION OF FOODS 

sugar), but saccharose (cane sugar) is sometimes present. 
Minute starch grains frequently occur in the sieve tubes; and 
in some plants the pores in the sieve plates are large enough 
for the smallest grains to pass through; but it is certain that 
no significant amount of carbohydrate circulates in this form. 

Oils can be absorbed into the phloem elements in the form 
of a fine emulsion, and in this form they can travel longitudi- 
nally through the sieve tubes and parenchyma cells. Chem- 
ical analysis shows, however, that very little non-nitrogenous 
food travels in this form, oil for the most part being trans- 
formed into sugar preparatory to translocation. 

The Propelling Power in Food Circulation. — The neces- 
sary conditions are always present for the circulation of foods 
in solution by diffusion throughout the length of the phloem, 
and foods must circulate to a certain extent in this manner ; but 
diffusion is a slow process and it alone can not suffice to carry 
foods fast enough when growth is rapid or when at the height 
of photosynthesis the leaves are taxing the utmost capacity 
of the conductive tissues. To illustrate the slowness of move- 
ment by diffusion alone: common salt in comparison with 
many other substances diffuses rapidly, yet in a 10 per cent, 
solution it required in one experiment 319 days to transfer a 
milligram of salt one millimeter, and under like conditions it 
would take fourteen years for egg albumin to travel the same 
distance. So we must assume that in plants diffusion is 
assisted in various ways, such as jarring due to the wind, etc., 
changes in temperature, circulation of the cytoplasm, and pos- 
sibly in other ways. We know that the contents of the sieve 
tubes are under pressure, for the tubes empty themselves when 
cut open, and this pressure would propel materials toward the 
places where for immediate use or for storage they are being 
removed from the tubes by the surrounding tissues. It is 
furthermore quite possible that effective conditions and forces 
are present of which as yet we have no clew. 



illustrative studies 177 

Illustrative Studies 
i. Study the phloem elements in the stem of squash, grape, 
and hop. Mount free-hand cross and longitudinal sections in 
dilute glycerine. Hunt for sieve tubes, companion cells, and 
phloem parenchyma. Find the perforations in the sieve plates 
in both cross and longitudinal sections. How far apart are 
the plates? and how many of them would food have to pass 
through in going I cm.? Draw sieve tubes from both points 
of view, showing the sieve plates. 

2. Study border parenchyma (page 45) and phloem ele- 
ments in cross and tangential sections of leaves. How far 
does the food have to travel from the palisade and spongy 
parenchyma before it can reach the conducting cells of the 
veins? Make drawings that will best illustrate what you 
have seen. 

Sections made from material imbedded in paraffin are best 
for this study, but good free-hand sections will do. To cut 
tangential sections of leaves free-hand coat thinly one end of 
a cork with melted rosin 2 parts and vaseline 1 part, and while 
this coating is yet warm press into it a piece of leaf that has 
lain for a while in 95 per cent, alcohol, first allowing the alco- 
hol to evaporate from its surface. With a little practice sev- 
eral good sections can now be cut from surface to surface. If 
preferred the sections may be cut on a sliding microtome. 

3. Cut some of the veins in leaves about sundown and 
darken them so that they cannot photosynthesize before they 
are examined the next morning, when they are to be bleached 
in hot alcohol and placed in an iodine solution. Does this 
reveal anything about the food-conducting function of the 
veins? An outline drawing colored lightly with a blue pen- 
cil where starch occurs will make a good record of this obser- 
vation. 

4. Girdle stems in the spring and note thereafter growth in 
diameter above and below the girdle. Study sections to note 
storage of food in both regions. 



CHAPTER XI 

STORAGE OF FOOD AND WATER 

Need of Food Storage. — As told in the preceding chapter, 
plants need food for growth and repair of tissues and to fur- 
nish energy to keep the life processes going. If food were 
being made continuously by the photosynthetic tissues day and 
night, and every day so long as the plant lives, and if the 
demand for food were uniform all of the time throughout the 
whole plant there would be no need of storing food for future 
use. But the actual conditions are quite the reverse of this. 

The photosynthetic tissues do not work at night nor through 
the winter and dry seasons after the leaves have fallen. And 
even when the leaves are on the demand for food is by no 
means the same at all times. More food is needed during the 
first part of the growing season when growth is most rapid, 
and more is needed when fruits are forming and advancing to 
maturity than at other times; and it is of great advantage to 
plants that they have the power and habit of storing food 
when the demand for it is relatively slight, for use when the 
need of it is greater. 

Trees and shrubs, denuded and entering upon their winter 
rest, are packed with reserve food in roots, trunk, and branches 
(Fig. 96). The greater part of this is kept till spring to sus- 
tain resumption of growth and the production of fruit, but 
some of it is necessary to sustain life during the winter. 
Every organism, so long as it lives, whether active or dormant, 
requires a certain amount of food to keep its life going. This 
is true even of dry seeds, although the amount consumed by 
them is so slight as to be difficult of detection; and parts in- 
tended for reproduction that become severed from the parent 
plant must first be stored with food for their nutrition until 

178 



THE KINDS OF STORED FOOD 



79 



they become independently established and begin to make food 
for themselves. 

The Kinds of Stored Food. — The best evidence of what 
constitutes the food of plants is found in seeds. What occurs 
stored up there must be plant food; 
and if we find, as we do, that the 
same stuff is stored in the branches, 
trunk and roots of mature plants, then 
we know that the food required for 
the embryo plant in the seed is the 
same as that needed by the adult 
plant. 

Both in seeds, bulbs, tubers, etc., 
and in the general plant body are 
found nitrogenous and non-nitrog- 
enous foods. Of the latter class 
starch, fats, and oils, are far the most 
common forms used in storage; 
glucose, lsevulose, and saccharose are 
much employed; and cellulose, inulin, 
glucosides, and mucilage less fre- 
quently. Different kinds of proteids 
and amides are the chief representa- 
tives of the nitrogenous class. 

Starch. — Starch occurs in the form of definite grains, either 
suspended in the cytoplasm or lying loose in the vacuoles. It 
is insoluble in the cell sap and is one of the most stable and 
permanent forms in which food is stored. The sizes, shapes 
and markings of the grains vary a great deal in different 
plants, and even in different parts of the same plant. The 
starch grains are, as a rule, much larger in special storage 
parts, as in fleshy roots and stems, and in the endosperm of 
seeds than in the ordinary stems and roots, and they range in 
diameter in different species from .002 mm. or less to nearly 
.2 mm. 

Starch grains in the special storage organs usually have 




Fig. 96. Camera-lucida 
drawing of tangential sec- 
tion through the wood of 
grape- vine, in, cells of med- 
ullary ray, and n, of xylem 
parenchyma, packed full of 
starch. 



i8o 



STORAGE OF FOOD AND WATER 




characteristic shapes and markings for the different kinds of 
plants. The forms are spheroidal, ovoidal, ellipsoidal, or poly- 
hedral where the grains crowd one another. Rarely rod and 
dumbbell shapes are found. The markings are in the form 
of concentric and excentric striations and more or less irreg- 
ular cracks. These characters are, 
in fact, so pronounced as to afford 
the means of detecting adulterations 
in ground and powdered food and 
drugs (see Fig. 97). 

The light and dark striations in 
the grains are layers of greater 
and less density, and are possibly 
due to periods of greater and less 
abundance of available sugar from 
which the starch is made. Some 
starch grains when partially di- 
gested, or swollen in a dilute solu- 
tion of potassium hydrate, appear as 
though made up of needle-shaped crystals radiating from the 
organic center of the grain, and it has been suggested that the 
denser layers of the grain are composed of denser groups of 
these crystals. The crystalline nature of the starch grain is 
also demonstrated by its behavior in polarized light as crys- 
tals do. 

Treatment of starch grains with boiling water seems to 
show that they are composed of two kinds of starch, one 
insoluble and the other soluble in boiling water, for the micro- 
scope reveals that the paste made in this way is not a complete 
solution and contains an abundance of undissolved remnants. 
The insoluble part is called a-amylose and the soluble /?-amylose. 
The percentage formula for starch is known (C 6 H 10 O 5 ), 
but the exact number of atoms to the molecule has not been 
definitely fixed. The number is tentatively expressed by mul- 
tiplying the percentage formula by n, thus, n(C 6 H 10 O 5 ). 
Starch grains are almost always colored blue with an iodine 






Starch from differ- 
ent sources. A, curcuma starch; 
B, corn starch ; C, tapioca starch ; 
D, rice starch, showing com- 
pound grains. 



THE KINDS OF STORED FOOD I 8 I 

solution; but when undergoing digestion they may. assume a 
reddish color with iodine due to the dextrines that have been 
formed from the amylose. In a few instances starch normally 
contains so much amylodextrin that it is colored red by iodine, 
as in the seed-coats of Oryza and Chelidonium. 

At the close of the growing season the amount of stored 
starch is at its maximum in ordinary stems and roots, and in 
special storage organs. The largest percentage of stored 
starch occurs, of course, in dry seeds, such as those of the 
cereals and legumes. Thus the potato is 20 per cent, starch, 
while beans contain 45, peas 58, oats 47, barley 48, rye 60, 
wheat and millet 64, maize, 65, and rice j6 per cent, of starch. 

Dextrose, Lcevulose, and Saccharose. — Dextrose (glucose or 
grape sugar) is the commonest form in which the non-nitroge- 
nous foods circulate throughout the plant. It is probably the 
form in which food is first made by the chloroplasts, and it 
arises secondarily by the digestion of starch, fatty oils, inulin, 
cellulose, cane sugar, etc. It occurs in solution in the cell sap, 
and when the sap is evaporated it is thrown down in the form 
of crusts or warty agglomerations of pseudo-crystals. Small 
percentages of dextrose frequently occur in storage tissues and 
sometimes in association with laevulose and saccharose. Dex- 
trose and lsevulose (fructose or fruit sugar) are the sugars 
in sweet fruits, in the nectar of flowers, and in the bulbs of 
Allium cepa and Ornithogallum arabicum, and in the under- 
ground parts of species of Primula and Globularia. 

Saccharose (cane sugar, beet sugar) occurs as reserve food 
in the maples/sugar- and sorghum-canes, beet-root, etc. Sugar 
maple sap yields somewhat less than 3 per cent, of sugar, 
sugar-cane sap about 18 per cent., sugar-beet sap 16 per cent., 
and sorghum-cane about 14 per cent, of saccharose. Cane 
sugar also occurs in some fruits such as the banana and 
pineapple. 

Fatty Oils and Fats. — Fatty oils are fluid at ordinary tem- 
peratures and the fats are solid. They are, of course, not 
soluble in, nor miscible with water or the cell sap; and in the 



,15 2 STORAGE OF FOOD AND WATER 

plant cell the fatty oils occur in a very fine emulsion, and the 
fats in groups of exceedingly minute crystals throughout 
the meshes of the cytoplasm. 

The fats and fatty oils as they occur in plants are mixtures 
of glycerine esters or glycerides of palmitic, stearic, and oleic 
acids; and the palmitic ester is called palmitin, the stearic, 
stearin, and the oleic, olein. Palmitin and stearin are solid at 
ordinary temperatures, and olein is fluid, and whether the mix- 
tures of these be fluid or solid depends upon their relative 
proportions. 

The fatty oils occur in greatest abundance in oily fruits and 
seeds, as in the fruit of the olive, and the seeds of castor bean, 
where 60 per cent, of the dry weight is oil, and in rape seed, 
with 50 per cent, of rape oil, and in walnuts, with 55 per cent, 
of walnut oil in the embryos. There are many seeds not classi- 
fied as oily which nevertheless contain enough oil to make them 
the source of its commercial production; some of the cereals 
are an example of this, and notably Zea mais. 

In reviewing all groups of seed plants it has been estimated 
that four fifths of them contain fats and oils as an important 
part of the non-nitrogenous reserve food in the seeds. 

The fats and oils in seeds are reserve food to be used during 
germination, but when they occur in fruits, as in the olive, 
they are not food for the embryo, but are useful in alluring 
animals to gather the fruit and scatter the seeds. 

Inulin. — Inulin is a carbohydrate occurring notably in the 
underground parts of some Compositae, such as Taraxacum 
and Dahlia, and it also occurs in the Campanulacese, Lobelia- 
cese, Liliacese, and Amaryllidacese, and a few other families. 

Inulin is soluble in the cell sap, and on freezing, or when 
placed in alcohol or glycerine, it is precipitated in the form of 
sphaerocrystals (Fig. 98). It is apparently made from dex- 
trose and lsevulose, and it is changed back into these or other 
sugars preparatory to its circulation. 

Reserve Cellulose and Amyloid. — Reserve cellulose is de- 
posited in the form of thickenings of the cell wall in the endo- 



THE KINDS OF STORED FOOD 



'33 



sperm of the date and 
other palms, and in spe- 
cies of Fceniculum, 
Strychnos, Coffea, Iris, 
Allium, Asparagus, etc., 
and in the cotyledons of 
some Leguminosoe and 
doubtless of some other 
plants. As in the case 
of inulin, dextrose and 





Fig. 99. Storage tissues of the cotyle- 
don of Impatiens Balsamina. A, from the 
resting seed, and B, from a germinating 
seed. In B the amyloid thickenings of 
the cell walls are partly digested away. 
(After Frank.) 



Fig. 98. Sphaero-crystals of inulin from tuber 
of Dahlia variabilis. A, precipitated from an 
aqueous solution; B, precipitated within the cells 
by long standing in alcohol. (After Sachs.) 



lsevulose are the materials 
from which reserve cellu- 
lose is formed, and when 
wanted for food it is trans- 
formed back to sugar until 
only a very thin primary 
wall remains (Fig. 99). 

Amyloid, like reserve 
cellulose, occurs as thick- 
enings to the cell wall of 
endosperm and cotyledons. 
It is colored blue with a 
solution of iodine, and in 
this respect is similar to 
starch. It is found in the 
cotyledons of Tropasolum 
and in the seeds of Im- 
patiens, Pseonia, and some 
Primulacese and Legumi- 
nosse. During germination 
amyloid is converted into 
sugar and dissolved away, 
leaving only the primary 
wall, as in the case of re- 
serve cellulose. 



184 STORAGE OF FOOD AND WATER 

Glucosides. — The substances embraced in this group may 
be nitrogenous or non-nitrogenous. They are characterized 
by yielding sugar and some aromatic and other compounds 
when decomposed by appropriate ferments, or when boiled in 
dilute acids or alkalies. They are bitter in taste, soluble in 
water, and may be isolated in crystalline form. Some of the 
glucosides undoubtedly serve as reserve food, but others may 
not be of use in this way. 

Amygdalin (CsoH^NO-n) is a glucoside occurring in bitter 
almonds. The enzyme emulsin, occurring with it, splits it 
into oil of bitter almond, prussic acid and glucose. Other 
glucosides are potassium myronate in mustard seeds, solanin 
in many Solanacese, such as bittersweet and Irish potato, sali- 
cin in the bark and leaves of willows, coniferin in the wood 
■and cambium of Conifers, digitalin, the poisonous substance 
in Digitalis purpurea, indican, occurring in species of Indi- 
gofera, which by its enzyme is broken down into a kind of 
sugar and indigo-blue. 

The full list of known glucosides would be a long one, and 
there is a multitude of bitter products in plants apart from the 
alkaloids, many of which will yet be found belonging to the 
group of glucosides. 

Mucilage. — This is characterized by its swelling enormously 
in water. It is known to be stored as reserve food in the 
tubers of some orchids and in the seeds of a few Leguminosse. 
It is clearly related to reserve cellulose in its chemical nature, 
and like the latter is transformed to sugar in its digestion. 

Proteids. — Proteids are the most complex of plant foods 
and at the same time the most important, since they are the 
chief constituent of the living protoplasm itself. Their exact 
chemical constitution has not been determined, but it is known 
that they contain carbon, hydrogen, oxygen and nitrogen, and 
many contain in addition sulphur, and a less number have 
phosphorus also. 

The food substances here enumerated before the proteids 
contain no nitrogen with the exception of some of the gluco- 



THE KINDS OF STORED FOOD 



185 



sides, and it is the proteids chiefly that contain the nitrogen 
supply of plants, 50 to 90 per cent, of the nitrogen in the vege- 
tative parts being held by proteids, and in seeds and spores 90 
to 98 per cent, of the nitrogen is contained in the proteid 
reserves. 

The proteids occur in three distinct conditions : as definite 
rounded granules known as aleurone grains, as smaller 
amorphous proteids, and as soluble proteid, which under nor- 
mal conditions is in solution in the cell sap. 

Aleurone grains occur most abundantly in seeds associated 
with starch or oil. In the garden pea and bean, for example, 
they occur as very small grains filling up the spaces between 
the starch grains, while in the castor bean they are much larger 




Fig. 100. To show aleurone grains. A, cells from cotyledon of seed of garden 
bean; n, aleurone grains; m, starch; B, cell from endosperm of castor bean; a, 
aleurone grain; I, ground substance; k, crystalloid; j, globoid. (A, after Sachs; B, 
after Frank.) 



and together with the oil fill up the fine meshes of the cyto- 
plasm (Fig. 100). Here and in some other oily seeds the 
aleurone grain is made up of a proteid body, called the ground 
substance, inclosing one or more proteid crystalloids, and one 
to several mineral granules called globoids, composed of a 
double phosphate of calcium and magnesium. 

Amorphous and soluble proteids occur in bulbs and tubers, 
and in the storage tissues of ordinary stems and roots. 

There are many different kinds of proteids in plants, but 
they are not yet well enough known to admit of complete 
classification. 



1 86 STORAGE OF FOOD AND WATER 

Most proteids are digestible in the animal body, and they 
are either soluble in water or made so by the enzymes pepsin 
and trypsin of the animal body or similar enzymes occurring 
in plants. Of these digestible proteids the globulins and albu- 
moses occur most abundantly in the reserve foods of seeds: 
the former of these coagulates on heating to 75 ° C, while the 
latter does not. Albumins occur in seeds and in the general 
cell sap and like the globulins coagulate on heating, but unlike 
them are soluble in pure water. Gliadin and glutenin, insol- 
uble in water but soluble in dilute alcohol, occur abundantly in 
the seeds of grasses. These give the sticky character to dough 
made from wheat and rye flour. 

All of the above-named proteids belong to the readily diges- 
tible class. Another class of proteids called nucleins are not 
so easily digestible, and some of them are apparently not at 
all so. These can be isolated by subjecting cells or tissues 
containing them to the action of pepsin and other proteid- 
digesting enzymes, since they remain intact after the other 
proteids have gone into solution. The nucleins are insoluble 
in water and dilute acids, but they dissolve readily in alkaline 
solutions. They invariably contain phosphorus and fre- 
quently iron, but not all have sulphur. They always occur 
in the nucleus, and possibly to a certain extent in the cytoplasm. 

Amides. — The amides contain carbon, hydrogen, nitrogen, 
and oxygen, and are simpler nitrogenous foods than the pro- 
teids. All amides appear to be soluble in the cell sap, though 
not with equal ease. They occur as reserve food chiefly in 
underground parts, such as fleshy roots, bulbs, tubers, and 
rhizomes; and in these places nearly the whole of the reserve 
nitrogen may be in amide compounds, such as asparagin, 
glutamin, tyro sin, and leucin. Of the nitrogen occurring in 
the beet root and potato, 30 per cent, and more is in the form 
of amides. Asparagin is the most common form in which 
nitrogenous foods are distributed throughout plants, and when 
seeds are germinating their proteids gradually are reduced for 
the most part to this form. 



PROCESS OF STORAGE I $7 

» 

The Process of Storage. — The leaves being the organs in 
which the non-nitrogenous, and apparently a good part of the 
nitrogenous, foods are made from the raw materials, the stor- 
age tissues, in whatever part of the plant, must get from the 
leaves in soluble and transportable form the foods which they 
are to store up. 

As has been said, the non-nitrogenous foods travel to the 
storage tissues chiefly as glucose, and the nitrogenous for the 
most part in the form of asparagin. Arrived at the storage 
tissues these relatively simple, soluble, and diffusible forms of 
food either accumulate there in the same form in which they 
came, or, as more often occurs, they are converted into more 
complex, less diffusible, or entirely insoluble forms. 

Glucose, for instance, may simply accumulate as glucose, as 
in the onion, or it may be condensed into the less diffusible 
saccharose, as in the root of the sugar beet ; or it may be con- 
verted into insoluble starch, as in the potato, or into oil, as in 
oily seeds. 

Asparagin may be stored as it is or first changed to some 
other amide such as leucin, tyrosin, and glutamin, as is the 
case in the roots, tubers, etc., of various plants. Or asparagin 
may be condensed into some form of proteid, in which case 
salts of sulphur and phosphorus may also cooperate. 

In these processes of transformation the various cell organs 
(nucleus, general cytoplasm, plasmatic membrane, plastids) 
play different, though not independent parts. Thus, the leuco- 
plasts make starch from glucose, and apparently the cytoplasm 
makes oil from glucose, and proteids from asparagin; and in 
all of this work it is almost certain that the nucleus lends a 
hand. 

We conclude that the leucoplasts make the starch because 
it first appears as a very minute granule within the leucoplastic 
body and gradually attains to its full size there. The leuco- 
plasts absorb the glucose and readjust its elements into the 
more complex starch. We note the fact but can not tell how 
it is accomplished, nor the steps in the process. As fast as 



188 



STORAGE OF FOOD AND WATER 




the glucose is thus taken out of solution more comes to the 
leucoplasts by diffusion, and the process advances until the 
leucoplasts are stretched to an almost or quite invisible film 
outside the starch grain. 

If the starch grain makes its beginning at the center of the 
leucoplast the successive layers are about of equal thickness 
all around and the grain becomes concentrically striated, as 
in the garden bean; but if the grain starts outside the center 
the additional layers are formed faster, 
and so become thicker, on the side of 
the greater amount of leucoplastic sub- 
stance, as we find them in the Irish 
potato (Fig. 101). 

Sometimes it happens that the starch 
begins to be deposited at more than 
one point in the leucoplast, so that two 
or more relatively small grains become 
closely associated, and adhering with 
more or less tenacity they constitute a 
compound grain (Fig. 101). 
In storage tissues, where simple grains are the rule, com- 
pound grains may also occur ; and in some instances, as in oats 
and rice, compound grains are the rule. In some cases small 
units of starch may begin to form a compound grain and then 
all become encased in a common starch sheath deposited by 
the exterior part of the leucoplast. These grains are called 
half -compound. 

The storage of oils and fats takes place in the vacuoles and 
throughout the meshes of the cytoplasm, and it may be in- 
ferred that the cytoplasm has more to do with their construc- 
tion and storage than have the other cell organs ; but the mere 
fact that reserve food occurs in a certain cell organ is not to 
be taken as evidence that other cell organs have not cooperated 
in its manufacture. 

In following the ripening of oily seeds it is found that 
sugars and starch are present in the immature seeds, but little 



Fig. io i. Showing con- 
centric and excentric stria- 
tions of starch grains, e, 
potato starch excentrically 
striated; f, compound starch 
grain from potato; g, bean 
starch concentrically striated. 



PROCESS OF STORAGE [89 

or no oil. As ripening proceeds, however, oil appears and 
gradually increases in amount, while the sugars and starch dis- 
appear, having unquestionably contributed the elements for 
the construction of the oil. 

In the leaves of Vanilla and other Monocotyledons have 
been found rounded bodies containing oil in their meshes, and 
these bodies have been supposed to be oil formers and have 
been named elaioplasts (Gr. claion, oil, and plassein, to form). 

Reserve sugar occurs in solution in the cell sap of the stor- 
age cells, and it probably exists within the cell wherever the 
cell sap penetrates. There is no special cell organ devoted to 
the storage of sugar, and whether the cytoplasm is most active 
in this process has not been determined. As has been 
stated, dextrose (glucose, grape sugar) is the most common 
form in which sugar circulates, and when it is to be stored in 
this form there is little left for the storage cells to do beyond 
taking it in and aiding in its accumulation after its concen- 
tration has become greater than in the surrounding tissues. 
Undoubtedly the plasma membranes in particular are active 
in this work, not only allowing but helping the sugar to accu- 
mulate when, governed alone by the recognized laws of dif- 
fusion, after its concentration in the storage cells equalled 
that in surrounding cells its continued entrance would be 
impossible. 

When the dextrose coming to the storage cells is trans- 
formed to saccharose, as in the sugar-beet root, the change is 
apparently advantageous to storage since saccharose is less 
diffusible and has less osmotic power than dextrose. 

It may be assumed that when sugar in solution is stored in 
cells the osmotic pressure within the cells becomes very great 
and saccharose would be more advantageous in high concen- 
trations since its osmotic pressure is only about half that of 
glucose. 

The reserve cellulose, amyloid, and similar thickenings of 
endosperm cell walls intended for food on the germination of 
the seed, as in the case of the date, Tropceolum majus, Impa- 



I9O STORAGE OF FOOD AND WATER 

tiens balsamina, etc., appear to be formed by the decomposi- 
tion of exterior portions of the plasma membrane, or by the 
more direct transformation of dextrose, which comes to. the 
cells abundantly while storage is going on. No special proto- 
plasmic organ for this work has been discovered, and it may 
be assumed that the exterior plasma membrane does the same 
work that it is supposed to do in the building of the ordinary 
cell wall (see page 5). 

It is noteworthy that in the construction of the cellulose, 
amyloid, etc., food reserves, the protoplast has accomplished 
without a special organ the same kind of chemical work as 
that done in the storage of starch where special organs, 
namely, the leucoplasts, are necessary. 

The formation of the proteid reserve foods can be followed 
to a certain extent where they appear as definite granules 
(aleurone grains), as in the seeds of castor oil and garden 
bean. In the early stages of development of these seeds no 
aleurone grains are present, but later minute projections from 
the meshes of the cytoplasm appear which gradually increase 
in size and fill up the interstices. The cytoplasm thus seems 
to be the immediate agent in the construction of the aleurone 
grains. Whether the cytoplasm breaks down its own sub- 
stance to form the aleurone or constructs this directly from 
amides, sugars, and salts of sulphur and phosphorus, has not 
been determined. It is possible, of course, that both methods 
are employed; but whatever the steps in the formation of 
reserve proteids, the simpler amides, sugars, etc., that are 
known to flow to the storage cells during the storage period 
contribute the necessary elements. 

Characteristics of the Storage Tissues. — Tissues pri- 
marily designed for the storage of food, as the endosperm of 
seeds and the bulk of the tissues in fleshy roots, tubers, etc., 
have relatively large cavities and thin cellulose walls, or where 
the walls are much thickened they have many thin places in 
the form of pits. Storage being a vital function the storage 
tissues are composed of living cells and when these cells die 



LOCATION AND EXTENT OF STORAGE TISSUES IQI 

they are no longer functional. Since most of the stored food 
must be digested before it can be moved from its place of 
storage the power to make digestive ferments or to carry on 
digestion directly without the aid of ferments must be one 
of the leading characteristics of the protoplasts of the storage 
tissues. When starch is to be stored the cells must be pro- 
vided with abundant leucoplasts. 

Location and Extent of Food Storage Tissues. — Food 
may be stored for longer or shorter periods in any living cell, 
but there are certain tissues which have the storage of food 
for their chief function. These are the endosperm and peri- 
sperm of seeds, and sometimes the mesophyll of cotyledons; 
the medullary rays and wood parenchyma of ordinary stems 
and roots, and of fleshy roots and stems where the rays and 
wood parenchyma greatly preponderate over the other tissues ; 
that portion of the pith immediately bordering the wood, and 
sometimes all of the pith; and the phloem parenchyma and 
thin- walled parenchyma of cortex and pericycle. 

The storage tissues in seeds make up their greatest bulk, 
and, unless the cotyledons are employed in storage, the embryo 
is only an insignificant part of the seed in size. When cotyle- 
dons that have been used for food storage are coming above 
the ground and turning green their mesophyll cells are deliv- 
ering up the stored food so that the cotyledons gradually 
become thin, and the mesophyll, through the secretion of 
chlorophyll by its leucoplasts (which then become chloroplasts) 
enters upon its new function of photosynthesis. 

The medullary rays are one of the most important of the 
storage tissues. Although the vast majority of the rays are 
very small, extending only a few cells vertically, and being 
one cell, or at most a few cells, wide tangentially, while they 
run to various distances towards the pith, they are yet so 
numerous that their bulk would sum up approximately 10 per 
cent, of that of all tissues of the wood. The smallest medul- 
lary rays are passed unnoticed by the naked eye, but these 
compose most of the medullary ray tissue. In a tangential 



9 2 



STORAGE OF FOOD AND WATER 



f 



section of wood one centimeter square where the naked eye 
distinguishes no medullary rays, the microscope reveals nearly 
three thousand, or there would be about thirty rays in a single 
square millimeter. These rays are thickly scattered amongst 
the other tissues and with great frequency come into con- 
tact with the tracheal tissues and wood parenchyma. The 
rule is that every ray touches one to many tracheal tubes, 
and when in longitudinal sections of 
wood a tracheal tube is examined along 
its length it is found that it comes into 
contact with about thirty rays for every 
centimeter (Fig. 102). These rays not 
only extend into the wood but also to 
greater or less distances out into the bark 
between the phloem bundles, and they are 
therefore in position to take on food as 
it travels from the leaves through the 
phloem, and to store it within them- 
selves, or to deliver a part of it to the 
wood parenchyma for storage. And when 
the period of storage is over, the rays 
are in position to deliver the food to the 
tracheal tubes for quick transportation to- 
wards the crown where unfolding buds and 
flowers and fruits are in need of it (Fig. 

94)- 

The medullary rays in the wood store non-nitrogenous foods 
in much greater abundance than the nitrogenous, and usually 
in the form of starch, and these ray cells must therefore pos- 
sess many active leucoplasts which seize upon the food as it 
comes in solution and change it to the insoluble condition 
before it, or more than a fraction of it, has a chance to enter 
the tracheal tubes and be swept back towards the leaves whence 
it came. It will be seen that the physical conditions would 
impel the dissolved food after entering the ray cells to pass 
on into the adjoining tracheal tubes by diffusion (see page 



Fig. 102. Camera- 
lucid a , low-power 
drawing of tangen- 
tial section of wood 
of Liriodendron tu- 
lipifera, showing fre- 
quency of contact of 
medullary rays with 
a tracheal tube. f, 
medullary ray; ^tra- 
cheal tube. 



LOCATION AND EXTENT OF STORAGE TISSUES 



193 



07), unless it were rendered insoluble, or the plasma mem- 
brane imposed its interdiction (see page 95). It may well 
be that the plasma membranes of the ray cells play an im- 
portant part in this way; but if they do they alter their 
behavior when growth is resumed 
in the spring", for they then allow 
the digested food to pass freely into 
the tracheal tubes (see Fig. 103). 

The phloem part of the medullary aI^ ^^^ ^H^/^T 
rays passes most of the non-nitrog- 
enous foods that come to it over 
to the xylem part of to the pericycle 
and cortex for storage, and reserves 
the bulk of the nitrogenous forms 
for itself. This fact stands sharply 
out when a cross-section of stem 
taken in autumn or late summer is 
placed in a drop of iodine solution. 
The part of the rays between the 
phloem strands is then colored yel- 
lowish or brown because of its pro- 
teid content, and the xylem part is 
blue or almost black, due to the 
abundance of its starch. 

The wood parenchyma, which is 
the predominant tissue in the xylem 
of many herbaceous plants, and 
occurs in greater or less abundance 
in the wood of trees and shrubs, 
assists the medullary rays in the the arr ° w s is being digested and 

- r 1 carried away. 

storage of non-nitrogenous foods, 

and almost the whole of fleshy roots and stems is composed of 

these two tissues, which there maintain the storage function. 

So long as the medullary rays and wood parenchyma remain 

living they retain the power and habit of storing food. In 




Fig. 103. Diagram to show 
food from the leaves descending 
through the sieve tubes and being 
stored in the medullary ray cells 
and xylem parenchyma, in A; 
and the digestion of the stored 
food and its ascent through the 
tracheal tubes when growth is re- 
sumed in the spring, at B. In 
both diagrams the black bodies 
indicate stored food; that at the 
points of the arrows is being 



194 STORAGE OF FOOD AND WATER 

trees that have heartwood and sapwood all tissues are dead in 
the heartwood. In other kinds of trees where heartwood is 
not formed the medullary rays and wood parenchyma may be 
alive from bark to pith, even in trees that are fifty or more 
years old. The rule is, however, that most of the stored food 
.occurs in the younger parts of the wood. 

The phloem parenchyma, like the phloem part of the medul- 
lary rays, stores up nitrogenous food reserves, and appar- 
ently for this purpose it is longer-lived than the other tissues 
of the phloem, living on sometimes for ten years or more when 
the sieve tubes and companion cells produced at the same time 
have long since died. 

The thin-walled parenchyma cells of the cortex and peri- 
cycle store up both nitrogenous and non-nitrogenous foods, 
and with them in this the collenchyma is often associated, and 
altogether they constitute a very significant part of the storage 
system. 

Fluctuations in the Solubility and Insolubility of Stored 
Food. — When the leaves of woody perennials have finished 
their work and shrubs and trees stand bare and apparently 
inactive it might be conjectured that their store of food would 
wait unaltered for the return of spring ; but this is by no means 
the case, for part of the food is rendered soluble and appa- 
rently is used in respiration throughout the dormant period, 
and the greater part may be changed from insoluble to soluble 
and back again as the outside temperature falls and rises. 
The maximum amount of starch is found in the fall, for a 
large percentage of starch in the bark is changed to sugar or 
oil during the winter, and in softwood trees and shrubs the 
same thing happens in the wood. In hardwood trees the 
change is not so great in the wood. A rise of temperature 
during the winter or early spring incites a change back to 
starch again. 

Digestion of Stored Food. — The forms in which foods 
are stored are suited as a rule to their safekeeping, but not to 
their circulation and use. Most foods are stored in an insol- 
uble form such as starch, oil, and the majority of proteids, 



DIGESTION OF STORED FOOD 195 

and only a few in their storage form are capable both of 
diffusion and assimilation, as glucose and saccharose. The 
chemical processes by which stored foods are made soluble, 
diffusible, and assimilable are called digestion. 

In carrying on digestion the protoplast usually employs to 
do this work a proteid body known as an enzyme or ferment 
which it has made apparently by a process of self-decomposi- 
tion that we call secretion; and of these enzymes the proto- 
plasts may possibly make as many kinds as there are varieties 
of food to be digested; and it is also possible that the proto- 
plasts sometimes incite digestion without the intervention of 
an enzyme. 

Free oxygen is necessary to the formation of enzymes, and 
these work best at warm temperatures ranging from 20 ° C. 
to 60 ° C. according to the variety; they are also more effective 
in the dark than in the light, since light, particularly of the 
violet end of the spectrum, tends to destroy them. 

It is not known just how the enzymes act in digestion. 
They incite the necessary chemical changes, but hold them- 
selves apart so that they are not themselves destroyed in the 
process. The result of this is that a small amount of enzvme 
can digest a relatively very large quantity of food, even up to 
100,000 or more times its own volume. 

Some of the many kinds of ferments produced by plants 
have been classified and named. Thus diastase converts 
starch into maltose (malt sugar). Malt as e converts maltose 
into glucose (grape sugar). Inulase converts inulin into 
fructose (fruit sugar). Invert ase splits saccharose (cane 
sugar) into glucose and fructose. Cytase changes cellulose 
to glucose. Pectase changes pectic substances in the cell wall 
to vegetable jelly. Emulsin and my rosin are representative of 
enzymes acting on glucosides and breaking them up into glu- 
cose and other substances. 

A group of enzymes known as lipases or steapsins split up 
fats and oils into fatty acids and glycerine. The enzymes, 
called proteolytic enzymes, that digest proteids are similar to 



196 



STORAGE OF FOOD AND WATER 








the pepsins and trypsins of the stomach and pancreas of ani- 
mals. The pepsins change proteids to the soluble peptones, 
and trypsins convert peptones or proteids directly into amido- 
acids. The trypsins are the more common of the proteolytic 
enzymes in plants. 

These enzymes occur in every cell where the food that they 
are fitted to digest is stored even transiently. In fact dias- 
tatic, inverting, and tryptic enzymes are so common that they 

seem to be a part of every 
protoplast. There are, how- 
ever, in special cases, cells 
or groups of cells devoted 
to the secretion of enzymes, 
as in the root of the horse- 
radish; and in the whole 
family of grasses the epi- 
dermis of the cotyledon 
secretes enzymes, which help 
to digest the reserve food 
in the endosperm, and the 
same thing occurs in the 
date palm (Fig. 104). 
Glands devoted to the secretion of enzymes occur in Dro- 
sera, Dionsea and Nepenthes (Fig. 105), but in these instances 
the enzymes do not act upon food stored within the plant, but 
upon insects, and the glands are comparable to those in the 
alimentary canal of animals. 

It seems that the enzymes do not reach the maximum 
amount in the cells until the process of digestion is well started, 
and it is for this reason that more diastase can be extracted 
from sprouted than from unsprouted barley. The mere pres- 
ence of the enzymes is, however, not enough to start digestion 
going; rather the impulse to grow and the actual inception of 
growth creating a demand for food seems to supply the stim- 
ulus that puts digestion in motion. Such procedure would of 
course be a vital one initiated by the living protoplast. 

Assimilation of Food. — We must keep in mind the fact 




Fig. 104. Enzyme-secreting cells of date 
cotyledon at A; and of cotyledon of Indian 
corn at B. The secreting cells are at e. 



ASSIMILATION OF FOOD 



197 




that the proteids, oils, starches, sugars, and other kinds of 
food have two distinct uses : to furnish chemical elements for 
the construction of the protoplasts and cell wall, and other 
special and useful products, such as nectar, aromatic oils, and 
enzymes, and to supply energy in the form 
and place needed to keep the vital machin- 
ery in motion. That food which is taken 
on by the protoplast and made a part of its 
own body is assimilated. We say that the 
food is lifeless but the protoplast is living. 
Necessarily the food passes from the life- 
less to the living condition by something 
that the protoplast does with it. In this 
process the food loses its identity. Sugar, 
oil, soluble proteids, etc., enter into new 
combinations resulting in the formation of 
protoplasm — the living substance. An at- 
tempt to explain how this most wonderful 
of transformations takes place would be 
the merest speculation. We know nothing 
profoundly about it. To begin with, we know very little 
about the sequence of steps in the formation of even life- 
less proteids from simpler substances, and protoplasm is sup- 
posed to be a complex aggregation of proteids, water, and 
other possible compounds. When we speak of life we have 
in mind a kind of energy manifested by the protoplasm alone. 
We may conceive of this manifestation as being due to a com- 
bination of certain rates, amplitudes, and paths of vibrations 
of the protoplasmic molecules or structural units. When new 
structural units are built from the food they would be suited 
to the same mode of motion as the others and they would 
presumably assume this mode because they are under the same 
conditions as the others. If this conception were true so far 
as it goes it offers nothing to clear up the great mystery of 
the creation of living from lifeless matter. That, however, 
the protoplasm has power to build its own substance by com- 



Fig. 105. Longi- 
tudinal section 
through a digestive 
gland of Drosera ro- 
tundifolia. (After de 
Bary.) 



I98 STORAGE OF FOOD AND WATER 

binations of different kinds of food and that the application 
of this power is self -regulating, resulting sometimes in growth, 
at other times in merely maintaining a balance between destruc- 
tion and construction, is satisfactorily established. 

Relation of Stored Food to Energy Supply. — The energy 
which plants draw upon to keep the vital activities going comes 
to them from the sun through the food. It seems that the 
facts are about as follows : The sun's energy is used by the 
chloroplasts to build carbohydrates. A part of this is decom- 
posed, yielding energy for the construction of proteids from 
carbohydrates and other substances. Other parts of the car- 
bohydrates and some of the proteids are oxidized, or broken 
down in other ways, yielding energy for the formation of 
protoplasm from proteids, etc. Some carbohydrates, proteids, 
and a part of the protoplasm itself are broken down and 
energy is thus set free for doing whatever the protoplast as 
a living agent has to do. 

This line of activities traces the source of its energy directly 
back to the sun. The food, as well as the protoplasm, has 
sun's energy stored up in it ready to be set free for doing 
work and subject to the direction of the protoplasts. The 
construction of food is in a manner similar to the winding of 
a weight, and when the food or the protoplast is decomposed 
the resulting readjustment of the elements keeps the vital activ- 
ities going, just as the falling of a weight may set and keep in 
motion a train of wheels, etc. More than 90 per cent, of the 
energy released in respiration appears as heat, and the remain- 
ing energy probably manifests itself for the most part in 
chemical reactions attendant on growth, repair, secretion, and 
other constructive processes. 

The Storage of Water. — The need of plants for special 
water-storage tissues is not so general as their need for tissues 
in which to store food. Under ordinary conditions water can 
be freely taken in from the soil which serves as the water res- 
ervoir for plants. But plants of desert regions or growing 
anywhere under xerophytic conditions (conditions making 
water hard to get, as when it is actually scarce or difficult to 



STORAGE OF WATER 



I 99 



absorb because of low temperature in the substratum, or be- 
cause there are substances in solution in amounts large enough 
to act as a poison or to retard the osmotic inflow into the 
roots) to which this reservoir is denied or more or less inac- 
cessible, have hit upon various compensating devices. One of 
these is the water-storage tissue. This is seen in its fullest 
development in succulent stems and leaves. In Mesembryan- 
themum Forskalii of the Egyptian desert approximately one 
half of each succulent leaf is made up of water-storage tissue 
(Fig. 106) ; and in epiphytic species of Codonanthe growing 
on a dry substratum nearly three 
fourths of the fleshy leaf is occu- 
pied by cells devoted to the stor- 
age of water (Fig. 81). The 
fleshy stems of cacti and some 
Euphorbiacese are largely com- 
posed of the same kind of tissue. 
In the leaves of Ficus elastica 
the protoderm of the upper sur- 
face divides tangentially and gives 
rise to several cell layers consti- 
tuting a typical water-storage tis- 
sue (Fig. 79). 

Water-storage tracheids some- 
times occur as terminals of the 
finer branches of leaf veins, as in Euphorbia splendens and 
Townsendia cespitosa (Fig. 107) ; and occasionally the meso- 
phyll cells have the characteristic wall thickenings of tracheids 
and apparently serve in water storage. The tubers of the 
potato and other fleshy underground parts serve for the stor- 
age of water as well as of food. 

Frequently, and especially in xerophytes, cells or groups of 
cells contain mucilage as a real cell content or as much thick- 
ened cell walls, the inner layers of which have become trans- 
formed from cellulose to mucilage. Mucilage has a great 
affinity for water, imbibing it with power and holding it with 




Fig. 106. Cross-section of leaf 
of Mesembryanthemum Forskalii 
showing a large part of the leaf 
devoted to the storage of water. 
a, water-storage cells; b, chloro- 
phyll-bearing cells; c, crystal of 
calcium oxalate. (After Schimper.) 



200 STORAGE OF FOOD AND WATER 

great tenacity. When the amount of mucilage is considerable, 
as is frequently the case in desert plants, such as the Aloes, 
cacti, certain species of Astragalus, and many others, it plays 
an important part in the water-storage function. 

Characteristics of Water-Storage Tissues. — The water- 
storage cells are characterized by having thin cellulose walls, 
or walls, if thickened, having many pits or thin places. These 




Fig. 107. Water-storage tracheids in the leaf of Euphorbia splendens. b, b, water 
storage tracheids; d, mesophyll cells; c, branch from a milk tube. (After Haberlandt.) 

cells readily imbibe water when plenty is at hand, and when 
the soil water is scarce they deliver their stores gradually to 
those tissues, such as the photosynthetic and meristematic, in 
which the scarcity of water would be most harmful to the 
well-being of the plant; and the water-storage tissues keep on 
distributing their stores until they themselves are wilted while 
the tissues to which they are tributory are maintained in a 
fresh and turgid condition. 

Illustrative Studies 
1. Cut free-hand sections of potato that has lain for some 
time in 95 per cent, alcohol to harden it. Mount the sections 
in a drop of water and after studying them with low and high 
powers let a drop of iodine solution diffuse under the cover- 
glass. Starch grains will be colored blue to black and pro- 
teids yellow. The cell contents can be seen to best advantage 
around the thinnest edge of the section, but there they will 
have dropped out to some extent and study of a thicker part 
of the section may be necessary to find out how densely packed 



ILLUSTRATIVE STUDIES 20 I 

are the cells with reserve food. Draw a few cells with their 
contents. Measure the starch grains. 

2. Study in a similar manner thin sections of soaked lima 
bean. Notice how the striations of the starch grains differ 
from those of potato starch. Notice the very distinct gran- 
ules of proteid stained yellow to brownish by the iodine. 
Draw a few cells with their contents, and measure the grains 
of reserve food. 

3. Cut with a dry razor free-hand sections of the endosperm 
of castor bean and transfer them to a watch glass containing 
95 per cent, alcohol 2 parts and castor oil 1 part and enough 
eosin to make a light red solution. After a few hours mount 
the sections in ricinus oil-alcohol without the eosin. This 
treatment will bring the aleurone grains out clearly and reveal 
their several parts (page 185) ; and it will show the grains to 
be imbedded in the meshes of the cytoplasm. Draw a few 
cells to show this, and on a larger scale a single aleurone grain 
in all its details. 

Put other sections for several hours in alcanna tincture 
(page 252) and mount them in a drop of dilute glycerine. 
The oil will be stained pink. Notice how it gathers in drop- 
lets of various sizes around the edges of the section, and the 
abundance of it in the uninjured cells. 

4. Test the endosperm of germinating the ungerminated 
castor bean for glucose (see under Copper Acetate and Fehl- 
ing's solution in Chapter XV). How do you interpret the 
results ? 

5. Examine with a high power in a drop of water starch 
from ungerminated and germinating barley. What evidence 
do you find that starch is digested during germination? 

Test both germinating and ungerminated barley for glucose 
by crushing the grains and boiling them in Fehling's solution. 

6. Cut free-hand or on a sliding microtome cross and longi- 
tudinal radial and tangential sections of grape stem. Mount 
the sections in glycerine-iodine (see under Glycerine and 
Iodine in Chapter XV). Note the extent of the storage of 



202 STORAGE OF FOOD AND WATER 

starch and proteids in phloem, medullary rays, and wood paren- 
chyma. Other woody stems will do, but the grapevine is 
especially fine for showing this. Make drawings from each 
region of a few cells with contents. 

7. Make thin cross-sections of rubber leaf and mount them 
in dilute glycerine. Note the clear tissue between the epider- 
mis and palisade cells. This is for the storage of water. 
Make a diagrammatic drawing of the leaf expressing the pro- 
portion of leaf devoted to water storage. 

8. Cut tangential sections (see Chapter X, page 177, par. 2) 
of a sunflower leaf and mount them in dilute glycerine. The 
large clear cells are water-storage cells. Draw a few of these 
cells with the surrounding mesophyll tissue. 



CHAPTER XII 

SECRETION AND EXCRETION 

Nature of Secretions and Excretions. — Secretions and 
excretions are distinguished from the reserve food told about 
in the last chapter by their evident uselessness in supplying 
materials and energy for growth, repair, etc. They there- 
fore, for the most part, remain practically unchanged in 
special cells or tissues, or are eliminated from the cells by 
excretion at the surface of the plant or into intercellular spaces. 
Sometimes they have a biological function, as in the case of 
nectar, and sometimes a physiological function, as in the case 
of enzymes, and organic acids excreted by the roots. 

By far the larger number of plant secretions belong to the 
class of ethereal oils and resins. These occur together, the 
resins dissolved in the oils and forming oleo-resins. When 
the oil evaporates, as it normally does in time, and can quickly 
be made to do on heating, the resins are left behind as a solid 
residue. 

The amount of resin present varies greatly, from the merest 
traces in many epidermal glands to more than 70 per cent, in 
some of the Coni ferae. 

The fragrance of these secretions is due to volatile sub- 
stances entering into the composition of, and even forming a 
very large part of, the oils, as, for instance, eugenol in clove 
oil, safrol in oil of sassafras, and cinnamon aldehyde in oils 
of cinnamon and cassia. 

Turpentine, produced chiefly by the Coni ferae, is the most 
abundant of the oleo-resin secretions. On distillation this 
yields oil of turpentine of commerce and resins varying in 
character with the different genera producing them. 

No sharp line can be drawn between secretions and excre- 
tions as these words have come to be used in physiological 

203 



204 



SECRETION AND EXCRETION 



literature. So long as the substance is in the cell that formed 
it we are apt to think of it as a secretion, but as an excretion 
when it is eliminated from the cell into intercellular spaces or 
at the exterior. The significance of these terms will be better 
apprehended by means of their particular application as we 
proceed. 

Secreting Cells and Glands in General. — Probably all 
living cells secrete digestive and oxidative enzymes, and all 
cells secrete their cell walls; but in certain numerous families 
of plants we find single cells or groups of cells called glands 
that carry on secretion as their special function. The secret- 
ing cells are sometimes descended from the protoderm, and 
therefore belong morphologically to the epidermis, and some- 
times they are descendants of the fundamental meristem and 
may occur in the cortex, pericycle, medullary rays and pith. 




Fig. 108. Glandular hair from the petiole of Pelargonium zonale. e, secretion 
from the globular gland on which it rests; B, portion of a cross-section through a 
nectariferous bract of Vicia sepium; n, nectar-secreting cells. (After Haberlandt.) 



Less frequently we find them descended from the procambium 
and belonging to the phloem or xylem parenchyma, as in the 
case of some of the resin-secreting cells of Conifers. 

There are three kinds of glands in regard to their location 
and form, namely, the superficial type which, descended from 
the protoderm, is borne at the outer surface and may rise above 
it in the form of hairs or scales; the interior globular type 



SKCKKTIXC CHLLS l\ GENERAL 



205 



of a more or less globular 
group of cells: and the interior tubular 
type in the f<>nn of a tube or canal. 
Glands belonging to the first type, com- 
monly known as glandular hairs, arise 
by the tangential division of a protoderm 
cell producing a multicellular hair, the 
apical cell of which enlarges and becomes 
the secreting cell (Fig. 108, A), or a 
group of secreting cells may compose the 
gland at the apex. Nectaries are usually 
of protodermal origin and their cells are 
frequently elongated radially in the form 
of papillae (Fig. 108, B) . 

The interior globular glands arise by 
the division of a cell 
usually of the 




or 
ground 



group of cells, 
meristem, and 



where these glands lie near the surface 



Fig. 109. Formation 
of an interior, globular, 
lysigenous gland of the 
leaf of Dictamnus fraxi- 
nella. A, g, g and c, 
mother-cells of the gland; 
c, from the protoderm, 
and g, g, from the fun- 
damental tissue. B, older 
stage where the cells 
have begun to form the 
secretion. The last stage 
is shown in Fig. 112. 
(After Sachs.) 



the protoderm may by 




cell division con- 
tribute cells to their formation (Fig. 
109) ; or sometimes the protoderm 
alone gives rise to the gland. Glands 
of the globular type are found in 
the clove, rind of orange and lemon, 
etc. (Fig. no). 

An intercellular cavity into which 
the secretions of the glandular 
cells are excreted is 
of two ways : The 
may split apart at 



formed in one 
secreting cells 
the center of 



the group and then draw- or grow 

away from the line of Separa- 
ble no. Cross-section through m 

a portion of orange peel showing tion, leaving an intercellular cav- 

the cavity of an interior, globu- •, /t^ - \ j_i j_* 

lar gland at g ; crystals of hes- ^ ( Fl §"' IIT ), 0r tlie Secreting 

pendin at h; calcium oxalate cells may break down altogether, 



crystals at k 
and Oesterle.) 



(After Tschirch 



leaving their secretions in the cavity 



206 



SECRETION AND EXCRETION 



formed by their disintegration (Fig. 112). The first method 
of forming the intercellular space is schizogenous, and the 
second, lysigenous. Sometimes the two methods are combined 
by the space beginning schizogenously and then being enlarged 




Fig. hi. Schizogenous resin duct in 
the young stem of ivy (Hedera helix), as 
seen in cross-section. A, early, and E, 
later stage in the formation of the duct. 
g, the mature duct; c, cambium; wb, 
phloem; b, bast fibers. (After Sachs.) 




Fig. 112. Lysigenous gland in 
the leaf of Dictamnus fraxinella. 
B, young gland, with cells begin- 
ning to secrete oil; C, mature gland 
where the secreting cells have 
broken down and left their secre- 
tion within the cavity thus formed; 
o, large drop of secreted oil. (After 
Sachs.) 



lysigenously, and to designate this method the term schizoly- 
sigenous is compounded. 

Interior glands of protodermal origin solely are found in 
Amorpha, Myrtus, Eugenia, Asarum, Croton, Crotonopsis, 
and some species of the Moracese, Urticaceae, Acanthacese, 
Saxifragacese, Crassulaceae, Geraniacese, and other families. 
By far the larger number of interior glands, however, are 
formed by the ground or fundamental meristem. 



SECRETING CELLS IN GENERAL 



20/ 



Interior tubular or canalicular glands are formed in essen- 
tially the same manner as the globular. A circular group of 
cells as seen in cross-section, and a long vertical row as seen in 
longitudinal section, forms an intercellular space schizogen- 
ously or lysigenously at its center throughout its length. If 
formed schizogenously the secreting cells form a sheath around 
the intercellular canal as seen in Fig. 113, and into this canal 



OQQQQPC I 





Fig. 113. Resin duct in leaf of Pinus silvestris, in cross-section at A, and in 
longitudinal section at B; h, cavity surrounded by the secreting cells; f, f, scleren- 
chyma fibers surrounding and protecting the duct. (After Haberlandt.) 

are excreted the secretions of the sheath cells. Fine examples 
of tubular canals are found in the needles and stems of pines. 

In the few instances where the tubular glands are differen- 
tiated from procambium strands they have the same method 
of formation as have those from the ground meristem. The 
tubular glands often branch and anastomose and thus form 
a complex glandular system. 

Laticiferous Vessels or Milk Tubes. — The milk tubes 
occurring in many families of plants form a much-branched, 
richly anastomosing system extending practically throughout 
the whole plant (Fig. 114). They have two methods of 
origin. In the Lobeliaceae, Cichoriacese, Papaveraceae, Cam- 
panulaceaa, Papayaceae, and a few Euphorbiaceae, and many 
Musaceae and Aroideae they are formed by cell fusions which 
take place early in the primary meristematic condition by 
digestion of separating walls. The anastomoses which unite 
the tubes into practically one system are formed also by cell 



208 



SECRETION AND EXCRETION 



fusions, or sometimes by outgrowth of branches from the 
tubes which push their way through intervening tissues and 

fuse with other tubes 
or branches. 

In the Urticacese, 
Asclepiadacese, Mora- 
cese, most Euphorbia- 
ceae, and Apocynacese 
each tube arises from 
a single meristematic 
cell which elongates 
and branches, keeping 
pace with the growth 
of the plant, and fus- 
ing its branches with 
those from other tubes 
and thus forming an in- 
tercommunicating sys- 
tem, so that when a 
wound is made the milk 
pours forth abundantly. 
The milk tubes re- 
main living for a long 
time and probably take 
an active part in the 
production of the very 
complex latex or milk, 
which may contain 
plastic or food sub- 
stances such as sugar, 
oil, starch, proteid, and 
aplastic or non-food substances such as tannins, alkaloids, 
some varieties of gum, caoutchouc, resins, and salts of cal- 
cium and magnesium. Some of these may be mere excre- 
tions of useless substances from other tissues, and some of 
them may be products of the tube itself destined for the useful 




Fig. 114. Laticiferous vessels from the cortex 
of root of Scorozonora hispanica. A, as seen 
under low-power, and B, a smaller portion under 
high-power. (After Sachs.) 



TANNIN CELLS 209 

purpose of healing wounds and giving immunity Erom para- 
sitic attacks, while others are clearly foods which find in the 
tubes an efficient means of distribution. 

Tannin Cells. — Tannin cells are found in various families 
of plants. They occur as single isolated cells or in small 
groups. The cells are approximately isodiametric or in vari- 
ous degrees of elongation. The longest known occur in the 
genus Sambucus, where they become twenty or more milli- 
meters in length and sometimes extend through an entire 
internode. 

Tannin cells are found in the epidermis, primary cortex, 
pericycle, phloem, medullary rays, and in the mesophyll of 
leaves. They occur in greatest abundance in the cortex and 
in the tissues of galls. Tannins seem to be by-products set 
aside in the tannin cells from the general circulation. It is 
uncertain whether the tannins are ever used to an appreciable 
extent in nutrition. They seem to be of service, however, in 
warding oft* parasites by their aseptic qualities and astringent 
taste. 

Special Enzyme-Secreting Cells. — In the Cruciferae, Cap- 
paridaceae, and a few other families are found special cells 
devoted to the secretion of enzymes, such as myrosin. The 
pungency of these plants is due to allylic mustard oil, pro- 
duced, it is said, at the moment of injury to the plant by the 
action of myrosin on the glucoside potassium myronate which 
is associated with the ferment. Glucose and potassium sul- 
phate are other products of this reaction. 

The digestive glands of insectivorous plants are unique in 
that their secretions digest animal tissues and are stimulated 
to activity by the presence of the captive. On the upper side 
of the leaves of Pinguicula are two kinds of glandular hairs, 
a long-stalked form secreting a sticky mucilage which holds 
fast the prey, and a short form, hardly appearing above the 
epidermis, which, when an insect is captured, secretes and 
15 



2IO 



SECRETION AND EXCRETION 



pours forth a digestive enzyme (Fig. 115). The short gland- 
ular hairs on the leaves of Dionsea muscipula behave like those 

of Pinguicula. 

In the pitchers of 
the genus Nepenthes 
unstalked digestive 
glands occur on the 
inside near the bot- 
tom. These pour forth 
an abundance of a 
mucilaginous diges- 
tive fluid the water 
a bundle of tracheids 




Fig. 115. Glands from Pinguicula. A, upper 
surface of leaf showing long-stalked gland at m, 
and short-stalked gland at n. B, cross-section through 
a short-stalked gland. {A, after Kerner, and B, 
after Haberlandt.) 



for which is supplied to the gland by- 
extending close up to the base of the gland. 

The most highly differentiated glands are found on the 
leaves of Drosera rotundi folia (Fig. 105). Here the gland 
proper, which is borne at the apex of a slender stalk, is com- 
posed of a bundle of tracheids surrounded by three layers of 
cells. The outer two layers seem to be especially concerned 




Fig. 116. Different forms of crystals of calcium oxalate. A, from the petiole of 
Begonia manicata. (After Frank.) 

in producing the secretion. The cuticle is permeable, and 
ordinarily an acid, sticky secretion is excreted at the surface 



SECRETION AND EXCRETION OF MINER \I.S 



I I 



in which the feet of insects alighting on the surface become 
entangled. The capture of an insect stimulates the "lands to 
secrete and pour forth more acid, and an enzyme similar to 
animal pepsin, by means of which the insect is digested. 

Secretion and Excretion of Minerals. — In some plants 
single cells, and strands or layers of cells forming a more or 
less extensive tissue, are devoted to the secretion and excretion 
of calcium oxalate crystals — excretion in the sense that while 
the crystals remain within the body of the plant and are con- 
tained within cells they have been set apart by themselves 
where, as a rule, their isolation continues throughout the life of 
the plant. The calcium oxalate crystals occur singly or in 
groups in a single cell, and either as simple or compound crys- 
tals, as shown in Fig. 116. The forms and association of the 
crystals may be influenced by the strength of the solution in 
the cell sap; but evidently 
the protoplast has a very 
important influence, for in 
certain cells and tissues only 
one kind of crystal may 
occur, as in the case of the 
acicular crystals in Trades- 
cantia, Pistia, Arisaema, etc. 

A secretion of relatively 
rare occurrence is that of 
calcium carbonate in thecys- 
tolith cells of some Moracese, Urticacese, and Acanthaceae, 
occurring commonly but not solely in the leaves. Following 
the development of a cystolith it is found that the outer wall 
of an epidermal cell, for instance, grows down into the cell 
cavity, swells out at the end and there becomes warty at the 
surface (Fig. 117). 

When a cystolith is treated with hydrochloric acid it quickly 
diminishes in size with the evolution of bubbles of C0 2 , show- 
ing that calcium carbonate is being decomposed. At the com- 
pletion of the reaction a skeleton of cellulose remains. The 





Fig. 117. Cystoliths from the leaf of 
Ficus carica. A, complete cystolith; B, 
cystolith from which the calcium carbonate 
has been removed for use in other parts 
of the plant. B, is from a leaf that had 
fallen off in autumn. (After Haberlandt.) 



212 



SECRETION AND EXCRETION 



cystolith is therefore a cellulose outgrowth of the wall infil- 
trated and encrusted with calcium carbonate. In nature the 
calcium carbonate of the cystolith comes and goes, and its 
secretion seems to be a method of accumulating it for use at 
some future time. 

The Process of Secretion. — Secretion is evidently a vital 
process, that is, it is carried on by the living protoplasm. The 
substances may be formed directly from foods, or from the 
disintegration of the cell wall, or from the decomposition of 
the protoplasm itself. It is possible that sometimes all of 
these methods are employed by a single cell or gland. The 
distinctive behavior of the cytoplasm of some secreting cells 
is evidence that it produces the secretion by self-decomposi- 
tion; for, preparatory to secretion, the cytoplasm becomes 
relatively very dense and granular, and as secretion sets in 
this density diminishes, until, when secretion stops, the cyto- 
plasm has become very much depleted. In some Rutacese the 
cytoplasm and nucleus of secreting cells disappear altogether. 

In some instances the 
evidence is very clear 
that the secretion has 
been formed from 
the substance of the 
cell wall, where the 
secretion appears just 
beneath the cuticle, and 
accumulating, pushes 
the cuticle off from 
the wall, as seen in 
Fig. 1 1 8. Secretions of mucilage and of ethereal oils and 
resins take place in this way. Of course these secretions do 
not necessarily come entirely from the substance of the wall, 
for it is possible that the protoplast makes a part of the secre- 
tion directly without first repairing the wall preparatory to 
decomposing it. 

The Excretion of Liquid Water. — On summer nights 





Fig. i i 8. Glands from the leaf of Ribes nigrum. 
A, young stage in the development of the gland 
where the cuticle is already being pushed up by 
the secretion, i. B, complete gland; k, secreting 
cells; h, cavity between the secreting cells and 
cuticle occupied by the secretion. (After Haber- 
landt.) 






EXCRETION OF LIQUID WATER 



" dew " hangs in droplets at the tips and along the edges of 
leaves of grass and many other kinds of plants. It was long 
supposed that in all cases these droplets were real dew formed 
from condensation from the atmosphere, but that this is by no 
means always the case is shown by chemical analysis of the 
drops, by the anatomy of the leaves where the drops occur, and 
by physiological experiment, as will now be set forth. 

Droplets from the leaves of In- 
dian corn on evaporation leave be- 
hind .05 per cent, of solid residue, 
from leaves of Brassica cretica 1 
per cent., and so on for different 
plants; and on incineration 15 to 
50 per cent, of this residue turns 
out to be ash. Real dew being dis- 
tilled water could not be expected to 
give such results. 

Sections through a leaf where the 
droplets occur are found under a 
microscope to have specialized cells 
or groups of cells having the ap- 
pearance of glands. These have 
been named hydatodes. One of the 
simplest of these is found on the 
upper and under surfaces of the 
leaves of Gonocaryum pyri forme, 
w r here each hydatode consists of a 
single epidermal cell differing from 
the rest in several details, as shown 

in Fig. 119. A portion of the outer wall grows out and forms a 
slender projection traversed longitudinally by a canal extend- 
ing from the mucilaginous apex to the cell cavity. The cell 
cavity is broad and funnel-shaped in its upper part, and again 
broadened below the neck of the funnel. As is the rule with 
secreting cells, the cytoplasm is unusually dense and the nucleus 
relatively large. In this instance water is absorbed by the 




Fig. 119. One-celled hydatode 
of Gonocaryum pyriforme, seen 
in cross-section at A, and from 
the surface in B. (After Haber- 
landt.) 



214 



SECRETION AND EXCRETION 




Fig. 120. Hydatode from the 
leaf of Phaseolus multiflorus. 
(After Haberlandt.) 



hydatode from surrounding epidermal and subepidermal cells 
and excreted through the narrow canal in the exterior pro- 
jection. 

On the under side of the leaves of Phaseolus multiflorus are 
curved hairs, as shown in Fig. 120, with outer walls thin and 

but little cutinized through which 
water filtrates and is excreted at the 
surface. 

In another class of hydatodes 
bundles of tracheids from the ter- 
minations of the vascular bundles 
supply water to water-excreting 
parenchyma cells or themselves 
excrete water directly into intercel- 
lular spaces. Hydatodes of this sort are not uncommon in 
ferns where they occur chiefly along 'the leaf margins. This 
type reaches its highest development where water-stomata are 
present in the epidermis through which water excreted into 
the intercellular spaces is expelled. This is illustrated by Pri- 
mula sinensis where the hydatodes 
occur in the teeth of the leaf margins 
(Fig. 121). 

Physiological experiments show 
that many hydatodes excrete water 
through the activity of their living 
cells, since excretion stops after they 
have been killed by brushing over 
with a solution of corrosive subli- 
mate, and they can not then be forced 
to excrete water even when the pres- 
sure in the tracheal tubes is made very 
great by connection with a U-tube 
with its long arm filled with mercury. 
The majority of hydatodes equipped 
with stomata will still excrete water, 
however, after they have been poi- 




Fig. 121. Radial longitu- 
dinal section through a hyda- 
tode from the leaf margin of 
Primula sinensis, i, upper, and 
j, lower epidermis; h, palisade 
cell; e, thin-walled paren- 
chyma, called epithem; g, in- 
tercellular space; f, guard cell 
of a water stoma; k, tracheal 
elements. (After Haberlandt.) 



EXCRETION OF LIQUID WATER 215 

soned, and by artificially increasing the pressure in the tra- 
cheal system excretion of water is hastened. 

The amount of water excreted by hydatodes is often remark- 
ably large. A young leaf of Colocasia nymphgefolia gave out 
48 to 97 cubic centimeters of water in one night, and a mature 
leaf of C. antiquorum excreted on the average 9 to 12 cubic 
centimeters nightly. 

The use of hydatodes seems to be that when transpiration 
is checked, as by the going down of the sun, while the absorp- 
tive activity of the roots is as yet undiminished, they may by 
the excretion of water prevent its filtration into the general 
intercellular system which should be kept open for the storage 
and circulation of the necessary oxygen and carbon dioxide. 
Hydatodes may take on other functions in special cases, such 
as the secretion of nectar or digestive enzymes. 

Illustrative Studies 

1 . Put small segments of pine branch into a saturated aque- 
ous solution of copper acetate, and after several weeks cut 
sections free-hand or on a sliding microtome and study them 
in a drop of dilute glycerine. Resin will be found stained an 
emerald green. Study longitudinal sections in the same way 
and draw the tubular glands from both points of view. 

2. Put a small handful of cloves into a widemouth bottle 
and pour water over them. Stopper the bottle with a per- 
forated cork and insert a U-tube flush with the lower surface 
of the cork. Let the long arm of the U-tube extend deep 
into another bottle. Set the bottle containing the cloves into 
a basin of water kept boiling, and the other bottle into a basin 
of cold water kept cold by running water or with bits of ice 
added from time to time ; this will condense the steam and oil 
that distils over. In this way the presence of volatile oil in 
the cloves can be demonstrated. 

3. Soften cloves in water, cut thin sections and mount them 
in a drop of strong KOH solution. Numerous oil glands 
will be found, and possibly needle-shaped crystals of potas- 



2l6 SECRETION AND EXCRETION 

sium caryophyllate formed by the reaction between the KOH 
and the clove oil. 

4. Make thin sections of lemon peel hardened in 95 per 
cent, alcohol and inclosed for sectioning in elder pith. Note 
the globular glands in different stages of formation. How 
close to the surface do they come? Do any of them show an 
opening at the exterior? Draw one of the glands with the 
surrounding tissue. 

5. With a sharp knife cut germinating date seeds cross- 
wise into slices about 2 mm. thick and put them through the 
process of fixing, hardening, and imbedding in paraffin, and 
sectioning, described in Chapter XIII. When it comes to 
staining the sections place the slide on which they are mounted, 
after the paraffin has been dissolved away in xylene and the 
xylene rinsed off with alcohol, in a dish of safranin (page 231) 
for a few hours, and then rinse out the surplus safranin in 
water, dehydrate quickly in 95 per cent, alcohol, rinse in xylene 
and mount in balsam. Study the epidermis of the enlarged 
cotyledon. These cells secrete enzymes for the digestion of 
the endosperm exterior to them. Draw a few of the secreting 
cells together with the adjoining cells of the cotyledon and 
endosperm, and by stippling indicate how the secreting cells 
differ in appearance from the others. 

6. Soak grains of Indian corn over night and cut the thin- 
nest possible free-hand sections across the embryo and endo- 
sperm. Mount the sections in dilute glycerine. Compare the 
appearance of the epidermis of the cotyledon with that of the 
date. These cells of the corn are also enzyme-secreting. 

7. Scrape hairs from the surface of Pelargonium zonale or 
other plant bearing glandular hairs, mount them in a drop 
of dilute glycerine and study them under high magnification. 
Draw some of the glandular hairs. 

8. Study longitudinal sections of the stem of some milk- 
weed or of the greenhouse Euphorbia splendens for laticifer- 
ous vessels. Make a drawing showing the branching and 



ILLUSTRATIVE STUDIES 2\"J 

anastomosing- habit of these vessels. Treat the section with 
iodine. Are there indications of starch and proteids^ 

9. Cut cross-sections through the cotyledons of acorns; 
examine them in water and allow a dilute solution of chloride 
of iron to run under the coverglass (see under Tannins in 
Chapter XVI). Note the indication of tannin in some of 
the cells. 

10. Cut cross-sections of the seed of Strychnos mix- vomica 
and test them for alkaloids as described under Alkaloids in 
Chapter XVI. 



CHAPTER XIII 
THE PREPARATION OF SECTIONS 

The preparation of thin sections of plant tissues is an abso- 
lute necessity in the study of plant histology not only that 
cell structure may be clearly seen but that the association of 
cells into tissues and the mutual relationship of the different 
tissue systems may be brought to light. Whether sections 
can be cut forthwith without special preparation of the subject 
to be sectioned depends upon the nature of the material and 
the particular question regarding it that is to be solved. The 
method of procedure to fit different cases will now be given. 

Cutting Sections Free-hand. — Good histological work 
can be done with some materials, such as the mature parts of 




Fig. 122. Manner of holding the razor and object in cutting sections free-hand. 

stems, roots, and leaves, by holding them between the thumb 
and forefinger of one hand while the section razor is wielded 
by the other (see Fig. 122). The forefinger is held hori- 
zontal and the razor rests upon it, being pushed from point to 
heel in cutting the section. There is never danger of cutting 
too thin sections by this method; rather, most of the sections 
are too thick, and skill comes only with much practice. Sup- 
pose a cross-section of a stem is being cut, it is not necessary 
that the section be complete, and the small but thin bits which 
one is sure to get in his efforts to secure thin sections are the 
most satisfactory under high powers. A very small segment 

218 



CUTTING SECTIONS FREE-HAND 2IQ 

of the stem usually suffices provided it extends from the sur- 
face to the pith. Tender, flexible parts, such as the blades of 
leaves, will need to be inclosed in elder pith before sectioning, 
and a good stock of pith should be kept on hand for this pur- 
pose. A piece of pith about an inch long is laid on the table 
and while held firmly between the thumb and fingers to keep 
it from cracking it is halved longitudinally with a sharp knife. 
If a leaf section is desired a strip of the leaf is held between 
the halves of pith while the section is cut through pith and 
all. Sections of delicate stems and roots and of buds and 
flowers may be made in the same way, only a groove should 
be made in the pith, of a size to hold the parts firmly enough 
while not crushing them. It is surprising how much really 
good work can be done with simple appliances of this sort. 

To get sections of the stone-cell tissue of nuts saw off as 
thin slices as possible with a hack saw and rub these down to 
the requisite thinness between two water hones kept wet. 
This is a slow process but it yields fine sections. A simpler 
way is to whittle off fine shavings with a very sharp knife. 
These shavings roll up and must be forcibly straightened out. 
They will break when this is done but the small bits will do. 

A sharp razor is a necessity to successful section cutting; 
and it is not sharp enough until it will clip a hair held so it 
is free to bend before the razor. A razor half hollow-ground 
on both sides is a good one for this purpose. The dealers 
offer razors ground flat on one side, but it is impossible to 
keep them sharp by the usual methods. A good shaving 
razor, so only the blade is not ground too thin, makes a suit- 
able section razor. 

While cutting sections keep the razor blade wet with about 
60 per cent, alcohol, and slide the sections into a dish of water 
before they have time to become dry. Never let sections 
become dry at any time, else they will shrivel and their cells 
will become filled with air which will prove a nuisance under 
the microscope. 

In studying stem and root structure three sections, each 



220 



PREPARATION OF SECTIONS 



from a different point of view, are necessary to an understand- 
ing of the character and extent of the different tissues ; these 
are a cross-section, a longitudinal section parallel to a medul- 
lary ray, known as a longitudinal radial section, and a longi- 
tudinal section at right angles to a medullary ray, called a 
longitudinal tangential section (Fig. 123). Good longitu- 
dinal sections are more difficult to get than cross-sections, but 
much of the difficulty is avoided if most 
of the surface is pared down so that 
only a small elevation is left to be sec- 
tioned, as shown in Fig. 124. It is a 
good plan to keep material that is to 
be sectioned in equal parts of alcohol, 
glycerine, and water; in this it may 
remain indefinitely, but only after several 
weeks will its best effects in softening the 
»:!?ffif j|l— J harder tissues and toughening the weaker 
be produced. 

Cutting Sections with a Microtome. 
— A simple form of microtome that 
can be clamped to the laboratory table is 
often of great advantage in cutting sec- 
tions with a razor (Fig. 125). If the 
material is hard enough to bear the 
strain it may be clamped directly in 
the jaws of the object holder by means 
of the thumb-screw S; or it may first 
be inclosed in elder pith, in velvet cork, 
or even in soft wood, before clamping 
in. The object is fed up a very lit- 
tle at a time by turning the milled 
head M of the micrometer feed-screw. The section razor is 
laid flat on the plate glass ways PP and pushed across the 
object with a long sliding motion from point to heel of the 
razor as shown in Fig. 126. In doing this the razor must be 
held firmly against the glass ways. After several sections 





Fig. 123. Showing the 
planes in which sections 
are cut, A, transversely; 

B, longitudinal radially; 

C, longitudinal tangen- 
tially. 



CUTTING SECTIONS WITH MICROTOME 



21 




have accumulated on the razor, which is kept wet with dilute 
alcohol, they may be swept with the finger into a dish of water. 
If it is desired to keep the sections in the serial order in which 
the}' were cut they may be transferred 
one by one into small phials, a single sec- 
tion to a phial. 

More elaborate microtomes have de- 
vices for carrying the section knife, or 
of holding the knife stationary while 
the object is made to vibrate back and 
forth against it, and in this w T ay sec- 
tions can be cut with increased rapid- 
ity and accuracy. In some forms there 
is an automatic feed which can be set to any desired thick- 
ness of section. One of the simpler forms of microtomes 
with a knife carrier is seen in Fig. 127, where the prin- 
ciple of its operation will quickly be recognized. As there 
shown the knife should be set at an angle to make a long 
sliding cut in all cases excepting where material imbedded 



Fig. 124. Showing 
how to trim a block for 
cutting longitudinal sec- 
tions. 




Fig. 125. Simple microtome for clamping to table. P, P, plate glass ways for the 
section knife; S, milled-head for clamping the object; M, micrometer milled-head for 
turning the screw that raises the object as the sections are cut; C, milled-head for 
clamping the microtome to the table. 



222 PREPARATION OF SECTIONS 

in paraffin is to be sectioned. In the latter event the knife 
is to be set square across at right angles to the direction 
of its motion, so that the sections are chopped instead of 
whittled off. In this way the edges of the sections adhere as 




Fig. 126. Showing the manner of holding the knife-blade on the glass ways, and, 
by the arrow, the direction of sliding the knife while cutting the sections. 

they are cut and form a ribbon which preserves the order of 
the series perfectly. In cutting paraffin sections with the slid- 
ing microtome of the type shown in Fig. 127 the knife needs 
to move through only a short distance each way, so the elbow 
may rest upon the table and the knife may be operated with 
a wrist movement merely. 

Care of the Section Knife^ — As has been said, and it will 
bear repeating, the section knife or razor must be kept sharp — 
what we call perfectly sharp, or as sharp as one can make it. 
The test is that it should clip a hair at a slight touch. If it 



CARE OF SECTION KNIFE 



will not do this it may need honing on a stone and then strop- 
ping on leather, or the stropping may be all that it needs. To 




tell what to do moisten the ball of the thumb and draw it 
lightly over the edge of the knife from tip to heel ; if the edge 



224 



PREPARATION OF SECTIONS 



gives the sensation of taking* hold of the skin throughout its 
length only stropping is needed, but if not, the knife must be 
honed. 

Before honing or stropping a microtome knife a steel back 
should be slipped on it so as to tip the edge to the proper 
angle, but an ordinary razor will not need this back. While 
honing hold the knife in the position shown in Fig. 128, keep- 
ing the back as well as edge against the stone, and while push- 









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Fig. 128. Showing the manner of honing the section-knife or razor. 

ing the knife edge foremost slide it at the same time from 
point to heel as shown by the arrow. Then turn the other 
face of the knife to the stone and repeat the stroke from point 
to heel towards the other end of the stone, and so on until 
the thumb test above described is satisfactory. Do not allow 
the stone to gum up; keep plenty of oil upon it if it is an oil 
stone, or if a water stone keep it well lathered with soap and 
water, and wipe the stone clean after honing. Then strop 



CYTOLOGICAL METHODS 



225 



the knife, drawing it over the leather back foremost from heel 
to point (Fig. 129), reversing the face for the back stroke, 
and keep this up until the knife readily clips a hair. 

Cytological Methods. — Within comparatively recent times 
methods have been worked out whereby the anatomy of cells 
and tissues can be laid bare in their finest details. These 
methods are intended first of all to preserve the structure of 
the protoplasts in its normal form, and then to cut a single 




Fig. 129. Illustrating how the section-knife or razor is drawn across the strop. 

cell into several sections while keeping these in their natural 
sequence, and finally to stain the sections so that different 
structures will take on different colors. 

The preservation of the. structure of the protoplasts is ac- 
complished by plunging the material into a solution, known 
as the fixative, which instantly kills the protoplasts so that 
decomposition incident to slow dying is prevented, and then 
hardening the protoplasts by transferring the material to alco- 
hol, beginning with weak alcohol and gradually increasing its 
strength until absolute alcohol is reached so as to avoid undue 
shrinkage. 

The material is next imbedded in paraffin, and sections 
adhering in ribbons are cut usually .005 mm. to .010 mm. 
thick, and these after mounting on a slide and being freed 
16 



226 PREPARATION OF SECTIONS 

from paraffin are stained with two or three different stains 
and then sealed in balsam in the form of permanent mounts. 

The processes thus briefly outlined will now be given in 
detail. 

The Fixing Process. — For the study of the finer structures 
of the protoplast Flemming's fixative has given on the whole 
the best results. The formula for this is : 

One per cent, chromic acid 16 parts, 

Two per cent, osmic acid 3 parts, 

Glacial acetic acid 1 part. 

Make the 1 per cent, chromic acid solution by dissolving 
1 gram of chromic acid crystals in 99 cubic centimeters of dis- 
tilled water, and dissolve 1 gram of osmic acid in 49 c.c. of 
distilled water to make the 2 per cent, solution. Then mix 
together 16 c.c. of the chromic acid solution, 3 c.c. of the 
osmic acid solution, and 1 c.c. of glacial acetic acid. Of 
course more of the fixative can be made so only the ingredi- 
ents are kept in this proportion. 

The osmic acid solution must be made with extreme care tc 
avoid all contamination with organic substances, which are 
sure to spoil it, as shown by the formation after a time of a 
black precipitate. For this solution procure a glass-stoppered 
bottle, wash it thoroughly with soap and water, rinse it many 
times, pour into it a saturated solution of bichromate of pot- 
ash in strong sulphuric acid, stopper the bottle and shake it 
vigorously, let it stand for a while &nd shake again, then pour 
out the solution and rinse the bottle again and again with dis- 
tilled water. Clean the stopper as thoroughly as the bottle. 
The osmic acid comes sealed in glass tubes and it is best to 
obtain it with one gram to the tube. Clean the outside of 
such a tube in the manner described for the bottle, stirring it 
about with a glass rod in the bichromate of potash solution 
and subsequent rinsings, and keeping the fingers off of it ; then 
guide it with the rod into the clean bottle ; pour into the bottle 
10 c.c. of distilled water, stopper the bottle and strike it against 
the palm of the hand until the tube of osmic acid is broken ; 



THE HARDENING PROCESS 227 

then pour in the remaining 39 c.c. of distilled water necessary 
to make the 2 per cent, solution. If the whole 49 c.c. of 
water were poured in at first it would have been more difficult 
to break the tube. Of course the distilled water must have 
been kept in receptacles free from organic matter. The fumes 
of osmic acid are hard on the eyes, nose, mouth, and lungs, 
and the face should be kept away from them. 

About twenty times as much Flemming's fixative should be 
used as of material to be fixed, and the material should be cut 
into pieces not greater than 2 mm. in any dimension, so that 
the fixative may penetrate quickly to all parts. It is conveni- 
ent to do the fixing in small phials, and if the material has a 
tendency to float it may be pushed under with a piece of filter 
paper that tightly fits the phial. Material should be fixed at 
once after it is gathered and if it grows at any distance from 
the laboratory the fixative should be taken along. 

Keep the material in the fixative for forty-eight hours and 
then remove it and pin it in little cheese-cloth bags which one 
can quickly make himself of the size wanted, and put these in 
running water for about six hours, or over night. If running 
water cannot be had then place the material in a bucket of 
water which is to be changed several times. 

A simpler and cheaper fixative which gives good results, but 
not quite equal to the above for dividing nuclei, is the chrom- 
acetic fixative. This is made by dissolving 1 grain of chromic 
acid in 99 cubic centimeters of distilled water and adding 0.5 
gram of glacial acetic acid. Use as described for the above 
fixative. 

The Hardening Process. — The material still kept in the 
bags is after washing placed in 20 per cent, alcohol for two 
hours, and then it is carried through a series of alcohols, each 
of the series 10 per cent, stronger than the one before it, 
remaining in each grade of alcohol for two hours until abso- 
lute alcohol is reached. If the material is not to be imbedded 
in paraffin at once it may be left in the 70 per cent, alcohol 
until needed, and then it may be carried on into the higher 
grades as if no interruption had occurred. 



228 PREPARATION OF SECTIONS 

The process of hardening may be considered complete when 
the 90 per cent, grade of alcohol has been reached, and the 
sojourn in absolute alcohol is intended to complete the dehy- 
dration of the material preparatory to its imbedding in paraffin 
or celloidin. In order to make dehydration more certain it is 
a good plan to have two bottles of absolute alcohol in each of 
which the material remains for two hours before it is trans- 
ferred to the solvent of paraffin or celloidin. 

The Process of Imbedding in Paraffin. — Transfer the 
material from the absolute alcohol to a phial containing equal 
parts of absolute alcohol and chloroform, and after two hours 
place it in a phial of pure chloroform, and again after two 
hours transfer it to another phial of chloroform, and in these 
instances enough chloroform to keep the material submerged 
is all .that is needed. Chloroform is a solvent of paraffin and 
the object now is to infiltrate the material with paraffin very 
gradually. Accordingly after two hours put a small shaving 
of paraffin into the last phial of chloroform where the material 
is, and shortly after this has dissolved add another shaving, 
and so on until the chloroform is saturated with paraffin at 
the temperature of the laboratory. All this while the material 
may have been left in the little bag of cheese-cloth for conveni- 
ence in handling, but now it should be taken out of the bag 
and laid back loose in the phial of dissolved paraffin. Place 
this phial on the top of a paraffin oven heated to the melt- 
ing point of the paraffin, which should be about 52 C. 
Remove the cork from the phial and let the chloroform 
evaporate. Add more paraffin a little at a time if needed to 
keep the material submerged. Keep the phial on the paraffin 
oven until the paraffin no longer has a sweetish taste, indicat- 
ing that all of the chloroform has evaporated. Make a small 
paper tray by turning up the edges of a piece of paper all 
around to the height of a centimeter and half fill this with 
melted paraffin heated hardly above its melting point, and into 
this pour the contents of the phial — paraffin and material. It 
is best to have the paper tray on something cold so that a crust 



PROCESS OF IMBEDDING IN PARAFFIN 2 2g 

of solid paraffin will quickly form at the bottom, and then with 
heated dissecting needles the material can be disposed in or- 
derly fashion over this crust, and when the paraffin has entirely 
hardened each piece of the material can be cut out with a good 
border of paraffin all around it. When the material has been 
arranged over the bottom crust blow upon the surface of the 
paraffin to harden it the more quickly, and plunge the tray 
into cold water as soon as the surface crust will bear this. 
The more quickly the paraffin is cooled the more firmly it sets 
about the material. The material may be left thus imbedded 
in paraffin until it is needed for sectioning. 

Sectioning Material Imbedded in Paraffin. — Tear off the 
paper tray and with a knife score deeply around the piece of 
desired material on both top and bottom surfaces, and then 
break the piece out. This will be called the paraffin block. 
Melt a piece of paraffin on the surface of the object carrier 
of the microtome. In the microtome shown in Fig. 127 the 
object carrier may be simply a piece of pine wood about a 
centimeter in cross-section which is to be clamped firmly in 
the jaws of the microtome. Before the melted paraffin on 
the object carrier has time to harden press into it the paraffin 
block, setting it up in the position to give sections in the de- 
sired direction; then pass a hot needle around the base of the 
block so as to fuse it thoroughly with the paraffin bed and 
make a firm union. Pare the sides of the paraffin block so 
that the opposing faces are parallel, and adjust the object 
carrier on the microtome so that the knife, standing at right 
angles to its line of motion will have its cutting edge parallel 
with the face of the block turned towards it. Now the sec- 
tions may be cut and they should adhere and form a ribbon. 
In cutting paraffin sections the knife does not need to be wet 
with alcohol or anything else as in other cases. If the paraffin 
breaks awa)^ from the material as the sections are cut the infil- 
tration may not have been successful, or the temperature of 
the room may be too low. If the sections crumple up as they 
are cut the room is probably too warm. The ribbons ought 



230 PREPARATION OF SECTIONS 

to be straight and if the front and back face of the paraffin 
block are trimmed parallel they are pretty sure to be straight. 
Sections seldom need to be cut thinner than .005 mm., and 
.010 mm. is a suitable thickness for most purposes. In mi- 
crometry the term mikron is applied to .001 mm. and the above 
thicknesses would be called 5 and 10 mikrons. 

Mounting Paraffin Sections. — The paraffin sections are 
made to adhere to the glass slips by means of albumin water. 
The stock solution of this is made as follows : Shake together 
equal parts of white of egg and distilled water and add to 
this a pinch of salicylate of soda to keep it from spoiling. 
For use add one drop of the stock solution to one ounce of dis- 
tilled water. This dilute solution will be referred to as albu- 
min water. 

Rinse thoroughly and wipe dry a glass slide that has been 
kept in a saturated solution of bichromate of potash in strong 
sulphuric acid. Place at the center of the slide a drop of albu- 
min water and with a dissecting needle drag the drop out in 
a thin film covering the space that is to be occupied with the 
sections. The albumin water should stay just where you put 
it without creeping away in the least. If it does creep the slip 
is not clean enough and it should be rinsed off and rubbed 
with a cloth moistened with alcohol. Cut the paraffin ribbon 
up into the desirel lengths and lay these on the film of albumin 
water, keeping the glossy side of the ribbon down, namely the 
side that was down on the knife after cutting, for this side 
adheres better to the slide than the other. When as many 
sections have been put on as will fill out under the cover-glass, 
or a less number if so desired, warm the slip over a flame until 
the ribbons lie perfectly flat and then draw away the albumin 
water with filter paper; at the same time keep the pieces of 
ribbon close together and properly lined up, and then place 
the preparation where it can dry for an hour or so at a tem- 
perature a little below the melting point of the paraffin. After 
this stand the slip on end in a dish of xylene to dissolve away 
the paraffin, and then in a dish of 95 per cent, alcohol to rinse 



STAINING THE SECTIONS 23 I 

out the xylene, and after this the sections will be ready for 
staining*. If the sections have been at all blackened by the 
osmic acid, as often happens, they should be bleached before 
staining'. To do this place the slide for a few minutes in a 
dish containing one part of hydrogen peroxide to twenty parts 
of 60 per cent, alcohol. 

Staining the Sections. — The finest results in staining are 
obtained with Flemming's triple stain, safranin, gentian violet 
and orange G, made by Griibler. Prepare the stains separately 
as follows : Make a saturated solution of safranin in 95 per 
cent, alcohol and dilute it with an equal amount of distilled 
water. Make a saturated solution of gentian violet in distilled 
water, to be used without dilution. Make a saturated solu- 
tion of orange G in distilled water and dilute it with five times 
its bulk of distilled water. Put the safranin and gentian 
violet in Stender dishes or tightly covered tumblers, and the 
orange G into a drop bottle. In addition to the stains have 
conveniently at hand : 

A drop bottle containing absolute alcohol. 

A drop bottle containing clove oil. 

A Stender dish or tumbler of xylene. 

A Stender dish or tumbler of 95 per cent, alcohol acidulated 
with a drop of concentrated hydrochloric acid. 

Proceed with the staining as follows : 

1. Stand the slide on end in the safranin for a few hours 
or over night. 

2. Remove the slide from the safranin, drain it, rinse it 
quickly in water, and set it on end in the dish of acidulated 
alcohol until the safranin stops coming off in clouds and the 
sections seem almost or quite decolorized. 

3. Rinse the slide quickly in water and set it on end in the 
dish of gentian violet for ten minutes. 

4. Remove the slide from the gentian violet, rinse it in 
water, hold it horizontally and flood the sections with orange 
G from a drop bottle for four seconds. 

5. Rinse off the orange G in water, drain the slide, and 



2$ 2 PREPARATION OF SECTIONS 

while holding it slightly slanting downward thoroughly dehy- 
drate the sections by having absolute alcohol flow over them 
from the drop bottle. 

6. Set the slide horizontally and flood the sections with 
clove oil from the drop bottle. This will gradually extract 
the gentian violet and the preparation should be watched under 
the lower power of the microscope so that this action may be 
stopped as soon as the gentian stain has lost its too great in- 
tensity and become transparent while yet distinct. Then drain 
off the clove oil and set the slide in the dish of xylene to thor- 
oughly rinse away the clove oil. 

7. Remove the slide from the xylene, drain it, place a drop 
of Canada balsam towards one end of the group of sections 
and lower a coverglass over it, beginning at the end where 
the drop of balsam is by bringing the coverglass into contact 
with the slide first at that end and gradually lowering it 
towards the opposite side so as to drive forward any air bub- 
bles that may become entangled with the balsam. Then set 
the slide where the balsam can dry for several days at about 
So°C. 

With this three-color stain the cytoplasm should be gray 
or brownish, the nucleus violet, the nucleolus red, cellulose 
walls uncolored or grayish, lignified, cutinized and suberized 
walls red. In dividing cells the chromosomes should be red, 
the spindle fibers violet, and the rest of the cytoplasm gray 
or brownish. 

Where a fine differentiation of the parts of the protoplasts 
is not so much sought after as a differentiation of the tissues, 
other simpler methods of staining may be used to good advan- 
tage. Double staining with cyanin and erythrosin gives ex- 
cellent results. For this are needed a saturated solution of 
cyanin in 95 per cent, alcohol, in a Stender dish or covered 
tumbler, and a saturated solution of erythrosin in clove oil, in 
a drop bottle. Set the slide on end in the cyanin for about 
ten hours, then rinse it in 95 per cent, alcohol until the cyanin 
no longer comes away in clouds, and this should require only 



IMBEDDING IN CELLOIDIN 233 

a few moments; then flood the sections for about four seconds 
with the erythrosin solution, drain and rinse thoroughly with 
xylene and seal in balsam. If the clove oil is not completely 
rinsed out in xylene the stains will fade out after a time. The 
time ratios in the stains will need to be varied for different 
materials. Iodine green may be used in place of the cyanin. 
It is cheaper than cyanin and is easier to work with. 

Cyanin and erythrosin can be used for sections cut free- 
hand or in any other way, and loose sections may be stained 
in watch glasses. A beautiful differentiation of the protoplast 
in shades of gray is obtained by Iron Alum-Hsematoxylin. 
Place the sections for two hours in a 3 per cent, aqueous 
solution of ammonia sulphate of iron, then wash in water 
for half an hour and place for about ten hours in a 0.5 per 
cent, aqueous solution of hematoxylin that has ripened in 
a bottle plugged with cotton, to let in the air, for two months. 
Remove from the hematoxylin, rinse in water five minutes, 
and place again in the iron alum to reduce the too intense 
stain. Keep watch of the bleaching process under the micro- 
scope until the parts of the protoplast appear no longer muddy, 
but still well defined. Now wash in water for an hour or 
more, and pass through alcohol and xylene, and mount in 
balsam. After the last washing in water the sections may, 
if desired, be very lightly counter-stained in a weak aqueous 
solution of fuchsin or orange G, and then, after again rinsing, 
be carried through alcohol and xylene for mounting in balsam. 

It must be borne in mind that always when sections in water or 
aqueous stains are to be mounted in balsam they must pass from 
the water into 95 per cent, or absolute alcohol for dehydration, 
and then into xylene which is a solvent of balsam. If it is 
found that the sections look milky or opaque when taken from 
the alcohol to xylene it is a sign that dehydration was not 
complete, the alcohol was not strong enough or the sections 
were left in it for too short a time. Opaque sections of this 
kind will clear up more or less after long standing in xylene. 

Imbedding in Celloidin. — Sometimes material that is not 



234 PREPARATION OF SECTIONS 

suitable for sectioning free-hand will also not give good results 
when imbedded in paraffin, on account of its size, hardness, or 
brittleness. In such cases we may get help in celloidin or 
collodion (gun cotton) for imbedding. The process of obtain- 
ing sections in this way is a slow one, and it is difficult to get 
sections as thin as ten mikrons. Therefore celloidin is to be 
looked upon as a last resort in a difficult situation. 

Material to be imbedded in celloidin is to be prepared in all 
respects as when paraffin is the imbedding material up to the 
90 per cent, alcohol in the dehydrating process. From this 
alcohol it is put into equal parts of ether and 95 per cent, 
alcohol (which we will call ether-alcohol) for several hours 
and then into a 2 per cent, solution of celloidin in ether-alcohol, 
where it should remain for several days and then be trans- 
ferred to a 5 per cent, solution of celloidin in ether-alcohol, 
whence after a few days it is to go into a 12 per cent, solution 
of celloidin, and after it has remained here a few days longer 
it is ready to be mounted on a pine block preparatory to being 
sectioned. 

Prepare a pine block large enough in cross-section to sup- 
port the material and with other dimensions adaptable to its 
being clamped in the object carrier of the microtome. Leave 
one end of the block rough and soak this end in ether-alcohol 
for a while and then dip it for a moment in the 2 per cent, 
celloidin solution. Remove the material from the thick cel- 
loidin and set it in right position on the prepared end of the 
block. Let the celloidin on the block stiffen for a moment 
only and then dip the celloidin end into the thick solution, 
remove it and hold it upright so that the new coating of cel- 
loidin may spread out somewhat over the end of the block 
and make a solid union, and as soon as the celloidin has har- 
dened a little at the surface drop the preparation into a dish 
of chloroform. After the celloidin has hardened in the chloro- 
form for a day put the preparation into equal parts of glycer- 
ine and 95 per cent, alcohol where it is to remain until wanted 
for sectioning. 



STAINING CELLOIDIN SECTIONS 235 

If it is more convenient to obtain ordinary collodion or gun 
cotton in place of celloidin it will do just as well as the latter. 

Sectioning Celloidin Material. — Clamp the block in right 
position in the object carrier of a sliding microtome (Fig. 
127), set the knife slanting so that a long gliding cut will be 
made, wet the knife with the alcohol-glycerine mixture, and 
proceed to cut the sections as thin as they can be made; but 
hope not for success if the knife is dull. With a camel's hair 
brush sweep the sections from the knife into a dish of 70 per 
cent, alcohol. 

Staining Celloidin Sections. — Transfer the sections from 
the 70 per cent, alcohol to the safranin solution described 
under the three-color method (page 231), and after ten hours 
or longer rinse them in 70 per cent, alcohol containing one 
drop of concentrated hydrochloric acid for every fifty cubic 
centimeters, until the safranin is very faint in the celloidin 
but still of sufficient intensity in the subject itself. Then pass 
the sections quickly through 95 per cent, alcohol and transfer 
them to a mixture of equal parts of bergamot oil, cedar oil 
and carbolic acid where they will be further dehydrated and 
cleared, and then they are ready for mounting in Canada 
balsam. 

Safranin and Delafield's hematoxylin combine well for 
double staining celloidin sections. To make the hsematoxylin 
solution dissolve one gram of hsematoxylin in six c.c. of abso- 
lute alcohol and add this drop by drop to 100 c.c. of a satu- 
rated aqueous solution of ammonia alum. Leave this exposed 
to light and air for one week, then filter it and add 25 c.c. 
each of glycerine and methyl alcohol and after five hours filter 
again. Let this ripen -for about two months before using. 

To double stain the celloidin sections place them in safranin 
for a day, rinse them in 50 per cent, alcohol, put them into 
hsematoxylin for about ten minutes, rinse them thoroughly in 
water and then in 35 per cent, alcohol and again in 50 per cent. 
alcohol; pass them quickly through acid alcohol (one drop 
hydrochloric acid in 50 c.c. of 70 per cent, alcohol), and then 



236 



PREPARATION OF SECTIONS 



put them through 70, 85 and 95 per cent, alcohols, leaving 
them about two minutes in each grade. Now clear the sec- 
tions for about two minutes in the mixture of equal parts of 
bergamot oil, cedar oil and carbolic acid, and mount them in 
Canada balsam. Here, as in all staining, the time ratios for 
the different reagents will need to be determined for different 
materials. 

Making Permanent Mounts in Glycerine or Glycerine 
Jelly. — Filamentous algae and fungi are pretty certain to 
shrink and become plasmolyzed when put through the process 
of mounting in balsam, but this danger can be easily avoided 
by mounting them in glycerine or glycerine jelly, preferably 
the latter. Fix the subjects in the chrom-acetic fixative de- 
scribed on page 227; wash them in running water for a few 




Fig. 130. Turn-table for cementing coverglasses to slides. For use where the mount- 
ing medium is glycerine or glycerine- jelly. 

hours and place them in a 0.5 per cent, aqueous solution of 
eosin for several hours; place for five minutes in a one per 
cent, solution of acetic acid in distilled water; wash out the 
acid completely in water and transfer the material to a ten 
per cent, solution of glycerine; tie a cloth over the dish to keep 
out the dust and allow the glycerine to concentrate by evapo- 
ration, and when it appears like undiluted glycerine place the 



STAINING THE SECTIONS 237 

material in a drop of glycerine on a glass slip, put on a cover- 
glass, wipe away all surplus glycerine with a moist cloth, dry 
the slide thoroughly, and with a turn table (Fig. 130) spin 
a ring of shellac cementing the coverglass to the slide. To 
make the shellac cement make a thick solution of ordinary 
gum shellac in 95 per cent, alcohol and add twenty drops of 
castor oil to the ounce. 

A more durable preparation is made by mounting in glycer- 
ine jelly. To make the jelly soak one gram of best gelatine in 
six grams of distilled water for two hours or so; add seven 
grams of pure glycerine and 0.15 gram of concentrated car- 
bolic acid ; heat this and stir it with a glass rod until it becomes 
clear ; put filter paper into a funnel, run distilled water through 
it, set it in an incubator or oven just warm enough to keep the 
jelly fluid, and filter the jelly into a bottle; cork the bottle. 
For use set the bottle in warm water until the jelly liquefies. 
To mount material in the jelly warm a slide, put a drop of the 
liquefied jelly on it and transfer the material from the concen- 
trated glycerine to the drop, put on the coverglass and after 
the jelly stiffens spin the ring of cement. 



CHAPTER XIV 

THE USE OF THE MICROSCOPE 

Adjusting the Microscope. — A type of microscope adapted 
to all histological purposes is shown in Fig. 131, where all the 
parts are plainly indicated. In lifting the microscope grasp 

-Oculaws 
Draw-tube-. 



Body Tube 

Noseptece 



Objectives ,£^ 



Upper Iris Diaphragm 
Suostage Rinj 

Condenser Mounting.. . 
lower Iris Diaphragm. 1 



ftack& Pinion 
Coarse Adjustment, 



Micrometer head or 
Fine Adjustment: 



Condenser Focusing Screw 
Mirror.. 



Mirror Fork 
tllfror Bar 




Horse ShoeDase. 



Fig. 131. Illustrating the parts of a compound microscope. 
238 



ADJUSTING THE MICROSCOPE 



39 



it by the pillar below the Stage, never by the arm or fine 
adjustment pillar, since the fine adjustment bearings might 
be injured in that way. Take a position near a window where 
the light from the sky will be unobstructed, but where direct 
sunlight will not fall upon the microscope. A north window 
is preferable where the too bright light from the sun cannot 
interfere with good vision. Place the microscope on the table, 




Fig. 132. Showing correct position at the compound microscope. 



as shown in Fig. 132, so that with the microscope erect you 
can look into the eyepiece without putting yourself in a con- 
strained position. The better microscopes have an inclination 
joint, so that the microscope can be inclined to suit the height 
of the observer; but since in histological work fluid reagents 
and mounting media are to be used it is better to keep the 



240 USE OF THE MICROSCOPE 

microscope upright and adjust yourself to it by adjusting the 
height of the table or chair. If the microscope is to be in- 
clined pull back on the fine adjustment pillar and not on the 
body tube. 

In nearly all histological work the object is seen by trans- 
mitted light; that is, by light that shines through the object, 
and this is accomplished by reflecting the light from below by 
means of the mirror, and where high powers are employed 
the light should pass through a condenser. The mirror has 
a plane and a concave surface, and where there is a condenser 
the plane surface only should be used, and without a condenser 
the concave mirror can in a measure fill its place, but it does 
not give so good results with high powers as does the con- 
denser. The condenser should have an iris diaphragm by 
means of which the light can be adjusted to the objective used, 
as will appear later on. 

For histological work there should be a medium and a high 
power objective, such as a f and a -J in. or a 16 and a 4 mm., 
mounted on a revolving double nosepiece, as shown in Fig. 
131. Assuming that the microscope is thus equipped revolve 
the lower power, that is, the § in. or the 16 mm. objective into 
position (in the figure the higher power is shown in position) 
and get ready some object for examination. To begin with 
there is nothing better than starch from the potato. To pre- 
pare this for examination a glass slip and coverglass obtain- 
able from dealers in microscope supplies will be necessary. A 
good supply of these should be at hand. Clean a glass slip 
thoroughly so that it is crystal clear when you look through 
it; lay it flat and put a small drop of water on it near the 
middle. Cut open a potato and with the point of a pocket 
knife scrape up a very small portion of the pulp not more than 
the size of two pin heads. Better take too little than too 
much. Put this in the drop of water on the slip. Clean a 
coverglass thoroughly and lay it over the drop. The cover- 
glass must be put on with some care else air bubbles will be- 
come entangled in the preparation, and they are a nuisance, 



ADJUSTING THE MICROSCOPE 24 1 

particularly to a beginner who may mistake them for some 
part of the thing he is studying. To avoid the air bubbles 
place one edge of the coverglass on the glass slip first and 
then gradually lower the coverglass while holding it between 
thumb and finger, pressing against opposing edges, and sup- 
porting it with a dissecting needle held under the upper edge 
as the coverglass begins to flatten out the drop of water. If 
now air bubbles are being entangled in the water the cover- 
glass may be rocked gently up and down with the dissecting 
needles until the bubbles are broken and driven out. When 
the coverglass is in position the water should fill out under it 
to the edges, but no more. If it runs out over the slip the 
drop was too big and the surplus should be wiped off with a 
piece of filter paper or clean cloth. Take note in this first 
preparation whether the drop was too large or too small so 
as to avoid the mistake in the future. The potato in the prep- 
aration should now appear as a very thin film. If it is in a 
lump it may be flattened out by pressing on the coverglass 
with the dissecting needle. Now place the slip on the stage 
under the clips (see the figure for the parts of the microscope 
as they are mentioned) and bring the preparation over the 
center of the condenser or to the center of the opening in the 
stage if there is no condenser. With the plane mirror, if 
there is a condenser, reflect light from the sky into the con- 
denser and adjust the mirror so that the preparation is seen 
to be illuminated while looking at it directly and not through 
the ocular. Then with the rack and pinion coarse adjustment 
run the body tube down until the lower power objective, which 
has been revolved into position, is within a quarter inch of 
the coverglass. Look in through the ocular and the field of 
view should appear circular and brightly illuminated; if it is 
not, adjust the mirror slightly until it is so. Now, still look- 
ing through the ocular, slowly rack the tube back until the 
object comes into view, and with the micrometer head of the 
fine adjustment, turning to right or left as seems to be neces- 
17 



242 USE OF THE MICROSCOPE 

sary, bring the object into sharp focus. The torn and crushed 
tissue of the potato will be seen and many very minute grains 
of starch which need to be seen with the higher power objec- 
tive. Accordingly draw the body tube up with the coarse 
adjustment and swing the high power objective into position, 
and then while watching outside the microscope run the body 
tube down until the objective all but touches the coverglass. 
It must be very close indeed, and you must hold your eye on 
a level with the stage to be certain about it and see that the 
objective does not press against the coverglass. Now look 
through the ocular and slowly draw the body tube up by turn- 
ing the micrometer head of the fine adjustment contra-clock- 
wise, until the preparation comes sharply into view. Starch 
grains of the potato have circular, excentric striations and if 
you can see these clearly with the high power it is an indica- 
tion that the microscope is adjusted to give a good image. 
If the striations cannot be seen you may be sure something is. 
the matter. See that the mirror is adjusted to give plenty of 
light and that the opening in the iris diaphragm is hardly more 
than one eighth inch in diameter, and see that the lenses of the 
ocular and the front lens of the objective are perfectly clean. 
These are the chief things to look to in securing a good image. 
If the lenses need cleaning breathe upon them and quickly 
wipe them off with a clean soft cloth; or dip the cloth into 
water and wash the lenses and then dry them with gentle 
pressure of a dry part of the cloth. A good tentative rule 
about the diaphragm opening is to have it about the size of 
the front lens of the objective in use. If there is then not 
enough light the opening may be made somewhat larger, but 
it will be seen that the image becomes less sharp the larger 
the diaphragm opening is made to be. Make it a rule from 
the beginning never to put up with a poor image. 

There are some details about the working of a microscope 
that need special mention. The tube length should be ad- 
justed to 1 60 mm. by sliding the draw tube in or out (see 
Fig. 131). When a revolving nosepiece is used the tube 



ADJUSTING THE MICROSCOPE 243 

length should be measured from the eyelens to the lower edge 
of the nosepiece. The coverglasses should be No. 2, except- 
ing when oil immersion objectives are employed with a work- 
ing distance so short as to require the thinner No. 1 cover- 
glasses. It is to be noted that the micrometer fine adjustment 
works through only a short distance, and therefore the coarse 
adjustment should always be used excepting for the finishing 
touches in getting a sharp image. If at any time the fine 
adjustment has been run to the extreme end of its range one 
way or the other the body tube should be run up with the 
coarse adjustment and the micrometer head of the fine adjust- 
ment should then be turned in the requisite direction to bring 
the fine adjustment to about the middle of its range. 

In some microscopes the condenser is provided with a focus- 
ing screw, by means of which the condenser can be raised or 
lowered to give the best results. Where there is a fixed 
mounting for the condenser the latter should be kept with its 
front lens flush with the upper surface of the stage. It is 
important that the lenses of the condenser should be kept 
as crystal clear as those of the ocular and objectives. 

For nearly all work the mirror bar should be kept parallel 
with the long axis of the microscope, so that the light will be 
reflected along the axis of the entire system of lenses. 

The clips (Fig. 131) are intended to press gently upon the 
glass slide so that it can be moved about over the stage steadily 
with gentle pressure of the fingers, but this cannot be done 
well unless the stage is kept clean. 

It has already been said that where there is a condenser the 
plane mirror should be used. The reason for this is that the 
condenser is made to bring parallel rays of light to a focus in 
the plane of the object, and if the concave mirror is used con- 
vergent rays would enter the condenser and be brought to a 
focus within the condenser system and the object would not 
be so well illuminated. 

Keep the fingers off from the surfaces of the lenses, for 
they are sure to leave a film that will need to be washed off. 



244 USE OF THE MICROSCOPE 

Most of the trouble which beginners experience in the use of 
the microscope comes from dirty lenses due to contact with 
the fingers or reagents, and to water or other fluids in which 
the object is mounted running out from under the coverglass 
and coming between the latter and the objective. These 
troubles are easily avoided with a little thoughtful attention. 
One can soon get into the habit of not touching the lenses with 
the fingers, and reagents will not run out from under the 
coverglass unless more than is necessary has been used. One 
should soon learn how large a drop of reagent is needed for 
a given size of coverglass. 

For bacteriological and cytological work an oil immersion 
objective will be necessary. A tV in. or a 2 mm. objective 
is best for general work in these lines. If such an objective 
is to be used it would be best to have a triple revolving nose- 
piece to carry the medium, the high, and the immersion objec- 
tive all at once. The use of the oil immersion objective is 
very simple. Put a small drop of cedar immersion oil on the 
coverglass directly over the object and run the body tube 
down with the coarse adjustment until the front lens of the 
immersion objective enters the drop and comes almost into 
contact with the coverglass. This is to be done while watch- 
ing the objective outside the microscope. Then while look- 
ing through the ocular draw the objective up with the fine 
adjustment until the object comes into focus. 

The higher the power of the objective the smaller the field 
of view, and when it comes to the oil immersions one sees but 
a very small area at once, and it is therefore much more diffi- 
cult to find a small object with them than with the lower 
powers. For this reason it is a good plan to find the object 
with a lower power and bring it to the center of the field, and 
then it will probably be in view when the immersion objective 
is used, but if it is not it cannot be far out of the way and a 
very slight movement of the preparation one way and another 
should suffice. With the oil immersion, when the object has 
been stained, the opening of the diaphragm may be made as 



DRAWING TO SCALE FROM MICROSCOPE 



245 



wide as is necessary to let in plenty of light. Indeed a good 
image can under these conditions be secured with a wide open 
diaphragm. When through with the oil immersion objective 
wipe it off with a clean soft cloth or with a piece of Japanese 
lens paper, and wipe off the coverglass in the same way. 

A very good artificial light for use with the microscope can 
be obtained with a Welsbach gas mantle and a balloon flask 
filled with a light blue solution of ammoniacal copper sulphate 
(Fig. 133). Dissolve a very small crystal of copper sulphate 
in enough water to fill the flask and add ammonia a little at 
a time until the solution loses all opalescence and becomes 
perfectly clear blue. Adjust the light and the blue condenser 
in front of the microscope until an image of the mantle, about 
natural size, falls on the mirror. Looking through the micro- 




Fig. 133. Method of illuminating compound microscope with gas lamp. C, balloon 
flask filled with ammonio-sulphate of copper; G, W r elsbach mantle. 



scope the light should appear white; if yellow, add more cop- 
per sulphate; if blue, dilute the solution. This light cannot 
be excelled and makes one independent of the weather condi- 
tions or the time of day. 

Drawing to Scale from the Microscope. — There are two 
w^ays of drawing from the microscope to a scale that can be 



246 



USE OF THE MICROSCOPE 




accurately determined; one is by the use of an eyepiece mi- 
crometer, that is, an eyepiece containing a glass disc with a 
fine scale etched on it. For purposes of drawing, a disc ruled 
off in very small squares is preferable (Fig. 134). In using 
this we need to know how large with a given objective an 
object would be that just fills out the space 
across one of these squares or divisions of the 
scale. This we can determine with a stage 
micrometer scale, which is simply a glass slip 
with a scale etched on it divided into tenths 
and hundredths of a millimeter. Put this in 
position on the stage and focus with the me- 
fig. 134. Glass disc d} um power and find how many of its small- 

ruled into squares to L J 

serve as a micrometer est divisions extend across one of the squares 

eye-piece and micro- - . . . ., . ., ~ 

area computer. This is or divisions on the eyepiece scale. Suppose 
to be placed in an or- ft ta k es fif tee n of them to do this, then we 

dinary eye-piece. 

know that any object that is found with the 
same objective to extend across one of the divisions on the eye- 
piece scale is exactly . 1 5 mm. in diameter. Try the high power 
in the same way, and of course the scale on the stage will be 
magnified more and a less number of its divisions than before 
will now cover a division in the eyepiece scale. If three of 
them now do this we know that any object under the high 
power extending across one division of the eyepiece scale has 
an actual diameter of .03 mm. An arbitrary but accurately 
determinable scale can now be fixed upon for drawing from 
the microscope. We may decide to draw 5 mm. long any- 
thing that covers one eyepiece scale under the medium power, 
in which case the magnification of the drawing would be 
T 5 5 = 26.6. This will serve to illustrate the method. When 
the eyepiece micrometer is ruled in the form of squares it can 
be conveniently used in determining the number of any par- 
ticular structures in a given area, as in a square millimeter. 
For instance, using the medium power with which an object 
.15 mm. in diameter would extend across the diameter of the 



DRAWING TO SCALE FROM MICROSCOPE 



^47 



square of the eyepiece scale, suppose we can count 9 stomata 
in the under epidermis of a leaf within one of these squares 
and we want to determine how many there would be in a 
square millimeter. That portion of an object which fills one 
of the squares would have an area equal to .15 X .15 mm. = 
.0225 sq. mm. How many times would this area have to be 
taken to make up one square millimeter? Of course the an- 
swer would be found by dividing 1 by .0225, which would 
give 44.4. Then the number of stomata in one square milli- 
meter would be 9 times 44.4 = 355. 




Fig. 135. Camera-lucida. M, mirror; P, opening to reflecting prism; K, knobs for 
regulating diaphragms that govern illumination from object and drawing paper. 



The other method of drawing to scale is carried out with 
a camera lucida, the most convenient form of which is shown 
in Fig. 135. The main structural details of this instrument 
are, behind the opening P a prism silvered on one of its sur- 
faces excepting for a narrow circular area at the center; and 
the plane mirror at M. Through the unsilvered part of the 
prism one can look and see the object; the mirror reflects the 
drawing paper placed on the table below it to the silvered sur- 
face of the prism and this reflects it into the eye. In this way 
the object and the drawing paper and the pencil held over the 
drawing paper are all seen at once superimposed; the object 



248 



USE OF THE MICROSCOPE 



appears spread out over the paper and with the pencil its out- 
lines can easily be traced- To use the camera lucida success- 
fully it is necessary to have some means of illuminating the 
object and drawing- paper with equal intensity, for if one 




appears brighter than the other they cannot both be seen with 
equal clearness. If the object, for instance, is too bright the 
point of the pencil cannot be accurately followed, and if the 
paper has the stronger illumination the pencil can be seen dis- 



DRAWING TO SCALE FROM MICROSCOPE 249 

tinctlv enough but the outline of the object becomes too dim. 
In the camera lucida of Fig. 135 there are two revolving dia- 
phragms with handles at K, with a series of openings, all but 
one of which in each series are covered with a graduated series 
of different intensities of smoked glass. With this provision 
one can decrease the light entering' the eye from the drawing 
paper or from the object until the object and the point of the 
drawing pencil can be seen with equal clearness. It is best to 
begin the adjustment with the free opening in both diaphragms 
in position, that is, with no smoked glass intervening between 
drawing paper or object, and then if the object, for instance, 
should be too bright the diaphragm relating to it can be re- 
volved until this is corrected, or if the drawing paper is too 
bright its diaphragm is to be revolved until the right degree 
of smoked glass is in position. 

In drawing from the camera lucida it is a convenience to 
have a drawing board (Fig. 136) adjusted to the same height 
as the microscope stage, and if the microscope is used tilted 
the drawing board should be set to the same angle as the stage. 
The mirror should be adjusted to bring the center of the 
drawing vertically below the middle point of the mirror, for 
the projection of the object off to one side of the mirror causes 
its distortion. 

The determination of the magnification of a drawing done 
with a camera lucida is made by projecting the scale of a stage 
micrometer upon the drawing paper by means of the camera 
lucida and drawing it there and measuring the drawing with 
a millimeter scale. Then the magnification is obtained by 
dividing the value of the magnified drawing by the actual 
value of the scale. For example, if one of the finest divisions 
of the micrometer scale (.01 mm.) measures 5 mm. in the 
drawing the magnification would be -q\ = 500. Of course 
the magnification would have to be determined for each ob- 
jective used and the tube length must be kept the same for the 
micrometer scale as for the object, and the distance of the 
drawing paper below the mirror must be kept the same. 



25O USE OF THE MICROSCOPE 

Use of the Polariscope. — The polariscope is very useful 
in detecting the presence of minute starch grains and crystals 
and in bringing out sharply fragments of sclerenchyma tis- 
sues in powdered drugs, etc. The polariscope consists of the 
polarizer which is placed beneath the stage, and the analyzer 
surmounting the eyepiece. Proceed with the polarizer as fol- 
lows: Adjust the mirror so as to reflect light into the micro- 
scope, and looking into the eyepiece rotate the analyzer. It 
will be seen that during this rotation through 360 ° the illu- 
mination of the field changes from brightness to blackness 
and back to brightness again. Turn the analyzer so that the 
field is at its brightest and place on the stage some potato 
starch mounted in a drop of water or dilute glycerine; these 
will then appear when looking through the eyepiece as though 
no polarizing apparatus were employed. Now turn the ana- 
lyzer through 180 or until the field is black and each starch 
grain will be seen to be traversed with a bright cross. It will 
be found that when thin sections of plant tissues containing 
crystals of calcium oxalate are treated in the same way some 
portions of them shine brightly out in the dark field, and the 
same thing is true of many sclerenchyma cells and fibers. It 
will be seen from this that the use of the polarizing apparatus 
may be of great service in identifying the parts of powdered 
foods and drugs where the fragmentary condition of tissues, 
cells, and cell contents increases the difficulty of finding out 
what the different parts are. 

The Use of Reagents on Microscopic Preparations. — 
When an object has been mounted in a drop of water under 
a coverglass the water can be replaced with other fluid re- 
agents without removing the coverglass or removing the 
preparation from its position under the objective. Suppose 
we wish to treat with a solution of iodine, starch from the 
potato that we have already examined in water: put a small 
drop of the iodine upon the glass slip close to but not touching 
the coverglass, and then with a dissecting needle or broom- 
straw drag the drop into contact with the coverglass, when it 



1 OF RE IGENTS 2 5 l 

will diffuse under and give to the starch the characteristic blue 
color which iodine is known to impart to it. The drop of 
reagent is not put at first in contact with the coverglass be- 
cause there would then be danger of its running over the top 
of the latter, wetting the front lens of the objective and pre- 
venting a good image. If this accident should ever happen 
in the use of a reagent there is nothing to do but to remove 
and w T ash the coverglass, and wash and wipe dry the objective 
lens. Sometimes it is desirable to hasten the replacement of 
the water under the coverglass by the reagent, and to do this 
it is only necessary to put a strip of filter paper in contact with 
the edge of the coverglass opposite the drop of reagent, when 
the water will run into the paper and the reagent will be drawn 
under the coverglass by capillarity. This process is called 
irrigation. It must be remembered, however, that very min- 
ute objects such as starch grains are pretty certain to be swept 
along in the currents, so that the slower process of diffusion 
should be depended on wherever practicable. 

Some reagents should not be diluted with water at all. 
Such, for instance, is chloroiodide of zinc. If a preparation 
already mounted in water is to be treated with such reagents 
the coverglass should be removed and all of the water drawn 
off with filter paper; then the reagent should be put on before 
the preparation has time to become dry. If acid reagents are 
to be used especial care must be taken not to allow them to 
get upon any parts of the microscope; if this should happen 
they must be washed off at once with plenty of water. The 
use of such acids as hydrochloric and sulphuric must be quickly 
over with, since their fumes are injurious to the eyes as well 
as to the microscope. 

When through with the microscope see that it is clean in all 
its parts and put it away under cover, where it will be free 
from dust. 



CHAPTER XV 

REAGENTS AND PROCESSES 

The different kinds of cell- walls and cell-contents may be 
demonstrated by the use of reagents which, in some cases, 
impart characteristic colors to walls and contents; in other 
cases act as selective solvents, dissolving some of the walls and 
contents, leaving others undissolved ; or the reagents may pro- 
duce precipitates the nature of which furnishes good evidence 
regarding the character of the substance which has united with 
the reagent to produce the precipitate. 

These reagents, together with their uses, will now be given 
in alphabetical order. 

Acetic Acid dissolves most ethereal oils, while most fatty 
oils are insoluble in it ; dissolves calcium carbonate with evolu- 
tion of C0 2 , while calcium oxalate is unaffected by it, and it 
therefore serves to distinguish between these two salts of cal- 
cium; solvent of crystals of hesperidin which have been depos- 
ited from the cell-sap of oranges, etc., when these have lain 
for some time in alcohol ; when various lichens are treated with 
it, crystals of calycin in acicular form are deposited after the 
lichens thus treated have been powdered and dried; I per 
cent, solution dissolves globoids in aleurone grains, while any 
crystals of calcium oxalate present are unaffected by it; when 
pieces of potatoes, carrots, etc., are macerated in it, the sepa- 
rate cells become isolated. Used in the preparation of various 
fixatives. 

Albumen. — The white of &gg is used with an equal amount 
of glycerine and a trace of salicylate of soda for fixing micro- 
tome sections to the glass slide, the sodium salicylate acting 
partly as an antiseptic. (Page 230.) 

Alcannin. — This is a coloring matter, obtained from the 

252 



ALCOHOL AMMONIUM VANADATE 253 

roots o( Alcanna tinctoria. A tincture of alcannin to be used 
as a reagent is prepared by placing alcanna root in 95 per cent. 
alcohol for about ten hours, or until a deep red solution is 
obtained, and then filtering off the solution and diluting it 

with an equal bulk of water. 

(1) Suberized and cutinized walls, when treated with a 
solution of alcannin for some hours, take on a pink color. (2) 
Alcanna tincture mixed with 1 per cent, glacial acetic or formic 
acid is used to fix and stain sections of elaioplasts from fresh 
material. (3) When sections containing fatty oils are treated 
with tincture of alcannin, the oil is colored pink. Sections 
containing ethereal oils and resins behave in the same manner. 

Alcohol. — The commercial alcohol obtained in this country 
is about 95 per cent, alcohol. In making alcohols from this 
of different strengths it answers all practical purposes to pro- 
ceed as if the commercial 95 per cent, alcohol were absolute — 
that is, very nearly 100 per cent. Thus, if 50 per cent, alcohol 
is desired, 50 c.c. commercial alcohol and 50 c.c. distilled water 
will give sufficiently accurate results for all histological work. 
If absolute alcohol is desired, it may be prepared by pouring 
the commercial alcohol over unslaked lime, and then distilling 
from this. Or better still, drive off the water of crystalliza- 
tion from copper sulphate by heating in an iron vessel. Put 
the powder into a bottle and pour 95 per cent, alcohol over it. 
Keep the bottle tightly stoppered. Water will be extracted 
from the alcohol and absolute alcohol results. 

Ammonium Molybdate. — A concentrated solution of am- 
monium molybdate in a saturated solution of ammonium 
chloride. This gives a yellow precipitate in sections contain- 
ing tannins. 

Ammonium Vanadate. — This is used as a test for solanin. 
The sections are treated with a solution prepared by dissolving 
1 part of ammonium vanadate in 1000 parts of a mixture of 
98 parts of concentrated sulphuric acid and 36 parts of water. 
If solanin is present, a yellow color appears, which merges 



2 54 REAGENTS AND PROCESSES 

into orange, then different shades of red, and finally into violet, 
and then all color disappears. 

Aniline Oil. — Excellent for dehydrating sections, since it 
will dissolve about 4 per cent, of water and may be kept dehy- 
drated by a small piece of solid KOH which is insoluble in it. 
The sections may be transferred from the aniline immediately 
into Canada balsam. 

Aniline Sulphate. — Make a saturated aqueous solution, 
As a test for lignified membranes mount the sections in the 
solution and add a drop of sulphuric acid, and a yellow color 
is given to the lignified membranes. 

Or pour sulphuric acid slowly into aniline oil until a pre- 
cipitate is produced throughout and then add water until the 
precipitate is dissolved. This will not require the addition of 
sulphuric acid to the sections. 

Balsam. — Canada balsam dissolved in xylol is, on the whole, 
the best medium for making permanent mounts of sections 
under a coverglass. For the method of doing this see page 
>233. Balsam in xylol can be obtained ready prepared of the 
dealers. 

Barium Chloride. — This is sometimes used to distinguish 
calcium oxalate from calcium sulphate. When barium chlo- 
ride is run under the coverglass, calcium oxalate, if present, 
is left unchanged, while a fine granular layer of barium sul- 
phate comes to incrust any crystals of calcium sulphate. (2) 
To determine the presence of tartaric acid, barium chloride 
and antimonic oxide in hydrochloric acid is run under the 
coverglass, producing, with tartaric acid, rhombic crystals of 
antimonium-barium-tartrate, whose obtuse angles measure 
128 . 

Benzol. — Used in detecting caffeine, thus : Sections are 
heated on the slide in a drop of distilled water until bubbles 
arise, then the water is allowed to evaporate, and the residue 
is dissolved with a drop of benzol. The benzol is then allowed 
to evaporate and the caffeine is deposited on the edge of the 
drop in the form of colorless needle-crystals. 



BERLIN BLUE BORAX-CARMINE 255 

Berlin Blue. — Useful in the study of the growth in thick- 
ness of the cell-membranes. In the study of marine algae - 
notably, C aider pa prolifera — it is used in the following man- 
ner : A vigorous alga is submerged for a few seconds in a 
mixture of one part of sea-water with two parts of fresh water 
in which has been dissolved sufficient ferrocyanide of potas- 
sium to give it the specific gravity of sea-water. The alga is 
then rapidly rinsed in sea-water and placed for about two 
seconds in a mixture of two parts of sea-water and one part 
of fresh water, to which has been added a few drops of freshly 
prepared ferric chloride. This produces in the membranes of 
the alga a precipitate of Berlin blue. The alga is then trans- 
ferred to sea-water for further growth. In case new lamellae 
are added to the membranes, the new portions will appear 
colorless, while the older portions will appear blue because of 
the Berlin blue which was precipitated in them. 

Bismarck Brown. — This is preeminently a nuclear stain. 
The powder is soluble with difficulty in water. It is a good 
plan to treat with boiling water and after a day or two to 
filter. Or a saturated solution may be made in 70 per cent. 
alcohol. Although Bismarck brown stains rapidly, it does not 
overstain. It may be used for staining in toto or for staining 
sections on the slide. 

Boracic Acid. — Used as a mounting medium for sections 
containing mucilaginous membranes. The sections are cut 
from dry material and placed in a ten per cent, solution of 
neutral lead acetate to harden the mucilaginous layers. Then 
the sections are stained in a solution of methyl blue, washed 
in water, and mounted under a coverglass in a 2 per cent, 
solution of boracic acid. The coverglass should be sealed 
down with a mixture of paraffin and vaseline, which is applied 
with a brush while melted. 

Borax-carmine. — A 4 per cent, solution of borax in water 
is made and to it is added 3 per cent, of carmine; an equal 
bulk of 70 per cent, alcohol is then added to this. The mix- 
ture is left standing for a day or so and then filtered. Sec- 



256 REAGENTS AND PROCESSES 

tions should lie in the stain for about twenty- four hours, and 
should then be transferred without previous washing to acidu- 
lated alcohol, made by adding four drops of hydrochloric 
acid to 100 c.c. of alcohol. Here they should remain until 
they become bright and transparent. This is a useful stain 
for aleurone grains, for differentiating cell-contents from cell- 
walls when the sections are subsequently stained with methyl 
green, and much used also in the differentiation of the cell- 
contents of filamentous algae. 

Bordeaux Red. — Used in conjunction with hematoxylin in 
staining nuclear figures, particularly where Heidenhain's pla- 
tinic chloride fixative has been used. The sections are placed 
in a weak aqueous solution of the Bordeaux until they are 
intensely stained; they are then rinsed and placed in a 2 to 
5 per cent, solution of ferric-ammonium sulphate for three 
hours. If the sections are mounted on a slide, they should 
be placed upright in this solution, so that any precipitate 
may not gather on the slide. Then the sections are care- 
fully washed in an abundance of water, and placed for twenty- 
four hours in a solution of hematoxylin prepared as follows : 
1 gm. of hematoxylin is dissolved in 10 gm. of alcohol and 
90 gm. of water. This is allowed to stand for about four 
weeks, and then an equal bulk of distilled water is added. 
The stain is then ready for use. When the sections are taken 
from the hematoxylin, they will be found overstained; they 
are, therefore, rinsed and placed in a 2.5 per cent, solution of 
ferric-ammonium sulphate, where they remain until examina- 
tion of the sections under the microscope shows the desired 
intensity of color. Then rinse in water 15 minutes, dehy- 
drate in alcohol, and pass through xylol for mounting in Can- 
ada balsam. 

Borodin's Method. — To determine the nature of a precipi- 
tate Borodin treats it with a saturated solution of the same 
substance as the precipitate is supposed to be. Thus, if the 



BROWN DISCOLORATION CARBOLIC ACID 257 

precipitate is supposed to be asparagin, it is treated with a 
saturated solution of asparagin. If the precipitate dissolves 
by this treatment, it is then some other substance than aspara- 
gin. Tin's method is not very reliable for substances which 
are very readily soluble, such as potassium nitrate. Care must 
be taken that the solution used for the test is entirely saturated. 

Brown Discoloration of Material in Alcohol. — Some 
plants, such as Monotropa, are apt to become quite brown in 
alcohol. This can be prevented by placing the fresh material 
in alcohol which is acidulated by vapor of sulphuric acid in 
the following manner: For each 100 c.c. of alcohol several 
cubic centimeters of concentrated sulphuric acid are poured 
over one half gm. of sodium sulphite, and the vapors aris- 
ing are conducted into the alcohol. This operation need 
require hardly more than a minute. After twenty-four hours 
the material should be transferred from the acid alcohol to 
neutral alcohol. Thereafter the material w T ill not discolor, and 
will take stains very well when used for histological purposes. 

Calcium Nitrate. — (i) Used to differentiate more clearly 
the lamellae of starch-grains. Potato starch, for instance, is 
placed in a rather strong aqueous solution of methyl violet. 
x\fter the grains have become deeply colored, they are treated 
with a weak solution of calcium nitrate, when the methyl violet 
becomes precipitated, particularly in the less dense lamellae of 
the starch-grains. (2) Calcium oxalate is precipitated in the 
form of crystals when sections containing oxalic acid are 
treated with a solution of calcium nitrate. The calcium nitrate 
is thus a test for the presence of oxalic acid. 

Canarin. — This is often used as a stain for tissues which 
have been cleared in caustic potash. Canarin is not affected 
by this reagent. 

Carbolic Acid (Phenol). — Used as a clearing agent. If 

leaves which have been hardened and bleached in alcohol are 

placed in three parts of turpentine and one part of carbolic 

acid, or in pure carbolic acid, the leaves will become so trans- 

18 



258 REAGENTS AND PROCESSES 

parent that their cellular structure may be made out from one 
surface to the other. Pollen-grains may be made transparent 
in the same manner. 

Carmalurn, Mayer's. — Carminic acid 1 gra., alum 10 gm. ; 
dissolve in 200 c.c. of hot distilled water; filter and add a few- 
crystals of thymol, or 0.1 per cent, of salicylic acid, or 0.5 
per cent, of sodium salicylate. This stains material well in 
bulk, with little danger of overstaining. If this happens, it 
may be corrected by washing with a 0.1 per cent, solution of 
hydrochloric acid. Material which has been stained in bulk 
with carmalurn may be sectioned, and the sections may then 
be double-stained with some aniline stain, such as blue de 
Lyon. See borax-carmine for another carmine stain. Very 
fine double staining may be achieved by placing sections first 
in an aqueous solution of iodine green and then for a some- 
what longer time in carmalurn. By this treatment lignified 
membranes are stained by the iodine green, while the unlig- 
nified membranes are stained by the carmalurn. 

Cedar Oil. — Sections which are to be mounted in balsam 
may first be examined in cedar oil .to determine their fitness 
for permanent mounts; if they are satisfactory, the cedar oil 
may be drained off and the balsam immediately added to the 
slide. Cedar oil has a clearing effect on sections which are 
treated with it. 

Thicker cedar oil with a refractive index of about 1.515 is 
used as an immersion fluid for homogeneous immersion lenses. 

Cedar oil is often used as an intermediary between alcohol 
and paraffin in paraffin-imbedding, but for plant tissues chloro- 
form is rather to be recommended. 

Chloral Carmine. — This is useful in clearing pollen-grains 
and staining their nuclei at the same time. It is prepared as 
follows: Carmine 0.5 gm. and .30 drops of officinal hydro- 
chloric acid (specific gravity, 1.13 or ij° B.) are added to 
30 c.c. of alcohol, and this is heated for about thirty minutes 
on the water-bath; then, after cooling, 25 gm. of chloral 
hydrate are added, and the solution is filtered until clear. 



CHLORAL HYDRATE — CHLOROPHYLL SOLUTION 259 

Chloral Hydrate. — Dissolve five parts of chloral hydrate 
in two parts of water. The chloral hydrate may be taken in 
grams and the water in cubic centimeters. This is one of the 
best clearing agents. Whole leaves, when boiled in this solu- 
clear quickly to such an extent that they may be studied 
by transmitted light throughout all of the cell-layers. Crys- 
tals in leaves may be plainly demonstrated in this way. This 
reagent is also very useful in clearing pollen-grains and em- 
bryos within the ovules. 

Chloral Hydrate-iodine. — Dissolve five parts of chloral 
hydrate in two parts of water and add enough finely powdered 
iodine to leave an excess undissolved after long standing. 
Shake before using. This is the best reagent for demonstrat- 
ing the presence of starch in chlorophyll corpuscles and in 
pyrenoids, or in any situation where the starch is surrounded 
and obscured by other substances. 

Chloroform. — Used as a solvent for fatty oils and of caro- 
tin. Used as a solvent for paraffin in the process of imbedding 
in paraffin. See page 228. 

Chloroiodide of Zinc. — Dissolve thirty grams of chloride 
of zinc, five grams of potassium iodide and 0.89 gram of 
iodine in 14 c.cm. of distilled water. Chloroiodide of zinc solu- 
tions should be kept in the dark. This reagent is one of the 
most generally useful in determining the character of plant 
membranes. By it cellulose walls are colored violet, lignified 
membranes a yellowish-brown, cutinized and suberized mem- 
branes from yellow to yellowish-brown. When sections con- 
taining sieve tubes are treated with chloroiodide of zinc and a 
rather weak solution of potassium iodide-iodine, the walls of 
the sieve tubes appear violet, while the pits in the sieve plates 
are a reddish-brown, due to the strands of protoplasm which 
penetrate them ; the callose plates are stained a reddish-brown. 
Mucilaginous walls are colored violet by this reagent. Chloro- 
iodide of zinc stains protoplasmic cell-contents from yellow to 
brown, and starch from purple to almost black. 

Chlorophyll Solution. — A freshly prepared strong solution 



260 



REAGENTS AND PROCESSES 



of chlorophyll in alcohol is used to demonstrate suberized and 
cutinized membranes. When sections are kept in the chloro- 
phyll solution for an hour or so in the dark, cutinized and sub- 
erized membranes are stained green, while lignified and cellu- 
lose membranes remain unstained. The chlorophyll solution 
will not keep, and should be freshly prepared whenever needed. 

Chromic Acid. — Solutions of i per cent, and 0.5 per cent, 
have been much used for fixing plant tissues. The material 
to be fixed should lie in the chromic acid for a day or more, 
according to the size of the pieces of material to be fixed. 
The material should then be thoroughly washed out in water 
and dehydrated by slow degrees in ascending grades of alco- 
hol (see page 227). A concentrated aqueous solution of 
chromic acid may be used as a macerating fluid to cause the 
separation of tissues into their separate cells. To this end 
rather thin bits of the tissue to be macerated should be placed 
in the chromic acid for about half a minute, and then carefully 
washed in water. This operation may be carried on with sec- 
tions under the coverglass. Silicious skeletons of diatoms, 
incrustations on the epidermis of Equisetum, etc., may be pre- 
pared by allowing the material to lie in concentrated sulphuric 
acid until it becomes black, and then, after transferring to a 
20 per cent, solution of chromic acid for some minutes, wash- 
ing thoroughly in water. In the case of Equisetum and the 
like the tissues should be scraped away from the inside down 
to the epidermis before treatment with the acids. Chromic 
acid is useful in the recognition of tannins, since sections con- 
taining tannins, when treated with a 1 per cent, solution of 
chromic acid, yield a brownish precipitate. 

Clearing Media. — See Carbolic Acid, Cedar Oil, Chloral 
Hydrate, Canada Balsam, Clove Oil, Eau de Javelle, Glycerine,, 
Origanum Oil, Turpentine, Xylol. A very successful method 
of clearing whole leaves is to boil them, if fresh, in 95 per 
cent, alcohol to extract the chlorophyll, place them in 5 per 
cent, hydrochloric acid for about ten hours, and then leave 
them until quite transparnt in a saturated solution of chloral 



CLOVE OIL — CORALLIN 26 1 

hydrate, hi the case of dried leaves the alcohol may be used 
cold. 

Clove Oil. — This is an excellent clearing medium, but it has 
the power of extracting certain stains, and so cannot be used 
in all cases ; it is, however, for this very reason of great advan- 
tage in the safranin-gentian violet-orange method of staining. 
See under this head. 

Collodion. — Used as an imbedding medium (see page 234). 

Congo-red. — This stain is particularly useful in studying 
the growth of membranes. Old membranes are, as a rule, 
left unstained by it, while the newly formed membranes are 
colored red. In a 0.0 1 per cent, solution — that is, 1 part of 
the stain to 10,000 of water — algae may continue to live and 
grow, and they are, therefore, well adapted to the study of 
the growth of membranes with the employment of this stain. 

Copper Acetate. — Used in the determination of tannins. 
Small bits of the plant to be tested are placed in a saturated 
solution of copper acetate, where they remain for eight or ten 
days; the sections are then placed on a slide in a drop of a 
0.5 per cent, solution of ferrous sulphate; after a few minutes 
the sections are washed in water, then in alcohol, and are finally 
treated with a drop of glycerine and examined under a cover- 
glass. This gives an insoluble brown precipitate with tannins. 

An alcoholic solution of copper acetate, to which has been 
added a small amount of acetic acid and glycerine, is used to 
demonstrate glucose in position within the cells where it 
occurs. The sections are laid in a mixture of the above solu- 
tion, and an equal volume of sodium hydrate in alcohol, and 
the whole is brought to boiling on the water-bath. Since glu- 
cose is insoluble in alcohol, the cuprous oxide which indicates 
the presence of glucose in this reaction is found to be depos- 
ited within the cells which contain the sugar. For other tests 
for sugar with a salt of copper see Fehling's Solution. See 
under Resin in next chapter. 

Corallin. — This stain is to be dissolved in a 30 per cent, 
or a saturated solution of sodium carbonate. It is particularly 



262 REAGENTS AND PROCESSES 

useful in staining the callose of sieve tubes. It is best to over- 
stain the sections and then to reduce the intensity of the color 
by immersing the sections in a 4 per cent, solution of sodium 
carbonate. 

Corrosive Sublimate. — See Fixatives. 

Cuprammonia. — This should be freshly prepared as needed 
in the following manner: Put copper filings into a bottle or 
flask, which is provided with a ground-glass stopper. Pour 
concentrated ammonia upon the filings and rock back and 
forth. Only sufficient ammonia should be used to cover the 
filings. When the solution will dissolve cotton, it is ready 
for use. This reagent is a solvent of cellulose. When sec- 
tions are placed in it for some time and are then rinsed with 
ammonia and finally with distilled water, crystals of cellulose 
are precipitated within the cells which are stained blue with 
chloriodide of zinc, and red with Congo-red. The crystals 
are again dissolved on the addition of cuprammonia. 

Cyanin. — This stain is almost insoluble in water, and should 
be dissolved in 50 per cent, alcohol. This is a useful stain for 
fats and all ethereal oils. Sections of fresh material, or mate- 
rial fixed in an aqueous fixative, such as an aqueous solution 
of corrosive sublimate or picric acid, will be sufficiently stained 
when left in the cyanin solution for about half an hour. Over- 
staining may be reduced with glycerine. The alcoholic solu- 
tion of cyanin, to which has been added an equal bulk of 
glycerine, is a good stain for suberized membranes, particu- 
larly after the sections have been treated with eau de Javelle, 
which destroys the tannins that prevent the membranes from 
taking the stain. When sections are placed in a dilute solu- 
tion of cyanin, — say 20 drops of a concentrated alcoholic solu- 
tion of cyanin in 100 c.c. of water, — for ten hours or longer, 
and are then washed in alcohol and placed in oil of cloves con- 
taining eosin, the lignified and suberized walls will be stained 
blue, while cellulose walls will be red. The sections may then 
be mounted in Canada balsam. When sections are placed 
for a quarter of an hour in a concentrated alcoholic solution 



DAHLIA DEHYDRATION 263 

oi cyanin, and are then washed in alcohol and transferred for 
a quarter of an hour to a 5 per cent, ammoniacal solution of 
Congo-red, the lignified membranes will appear blue, while 
the unlignified membranes will appear red. After washing in 
alcohol and afterwards in xylene, such sections may be mounted 
in Canada balsam. See also page 232. 

Dahlia. — An aqueous solution of from 0.00 1 per cent, to 
0.002 per cent, is used for staining live nuclei. The dividing 
nuclei of Tradescantia Virginica, for instance, when kept in 
this stain for a few hours, become weakly stained. The struc- 
ture of pyrenoids is well demonstrated by fixing them in equal 
parts of a 10 per cent, solution of potassium ferricyanide and 
a 55 per cent, solution of glacial acetic acid and then staining 
with dahlia, and finally swelling the pyrenoids somewhat in a 
weak solution of potassium hydrate. 

Decalcification. — Three per cent, of nitric acid in 70 per 
cent, alcohol is a good decalcifying reagent. The material 
should be left in the solution for several days. Chromic acid 
has a decalcifying action; a 1 per cent, to 2 per cent, solution 
should be used, and the material should be left in this until 
decalcification is found to be complete. 

Decolorizing. — Material which has become brown in alco- 
hol may be decolorized in the following solution: To each 100 
c.c. of alcohol is added from 0.2 to 0.5 c.c. of concentrated 
sulphuric acid and as much potassium chlorate as can be trans- 
ferred on the point of a knife. The material is to lie in this 
solution for eight or ten days, and is then to be transferred to 
alcohol or to equal parts of alcohol, glycerine and water for 
preservation. See also under Brown Discoloration, etc. 

Dehydration. — This is best accomplished by cutting the 
material into as small pieces as is practicable, and then placing 
it in 20 per cent, alcohol, and then into ascending grades of 
alcohol of 10 per cent, increase at intervals of about two hours. 
Microtome sections mounted on the slide may be transferred 
to strong alcohol without injury. In passing from water or 
aqueous stains to Canada balsam, the material should first 



264 REAGENTS AND PROCESSES 

come into strong alcohol, and then into xylol to insure com- 
plete dehydration, and to infiltrate the material with a solvent 
of balsam — namely, xylol. Aniline is also a good dehydrat- 
ing agent. The preparations may pass directly from water 
into the aniline and from the aniline into the balsam. A stick 
of potassium hydrate placed in the aniline will keep the latter 
dehydrated. Potassium hydrate is not soluble in aniline. Very 
thin microtome sections which are found not to be injured by 
drying may be allowed to dry, and then may be placed in xylene 
and thereafter transferred to balsam. See page 227 for further 
description of the process of dehydrating. 

Desilicification. — This is accomplished by hydrofluoric acid. 
A glass vessel is coated on the inside with melted paraffin to 
prevent the action of the acid on the glass. Alcohol is then 
poured into the vessel and the material is immersed in the 
alcohol; then the hydrofluoric acid is added, drop by drop. 
The process should be completed in a few minutes. Care must 
be taken not to breathe the fumes of the acid, since they attack 
the mucous membranes. 

Diastase. — This may be prepared as follows : Germinate 
barley in the incubator between pieces of blotting-paper until 
the plumule has reached a length of about 2 mm. ; then dry 
the barley on the water-bath and grind to a fine powder. 
When a diastatic solution is desired, pour over 10 gm. of the 
powdered barley 1 liter of water containing 2 c.c. of chloro- 
form ; let stand for ten hours at about 1 5 ° C. and filter. The 
water filtered off will contain the diastase in solution. Add a 
little chloroform and preserve in a dark place. Starch-grains 
may be mounted in this solution under a coverglass and kept 
from drying in a moist incubator, and the effect of the diastase 
on the starch may be studied from time to time under the 
microscope ; or a 1 per cent, starch-paste may be made to which 
about an equal amount of the diastatic solution may be added, 
and then at intervals samples from the mixture of starch and 
diastase may be tested with a solution of iodine. The starch 
will, after a time, be changed into dextrines and grape-sugar, 



DIGESTIVE FLUIDS EAU DE JAVELLE 265 

and will no longer give a blue color when tested with a solution 
of iodine. 

Digestive Fluids. — To remove from sections aleurone 
grains which are so numerous as to obscure the nucleus, the 
sections should he treated for twenty-four hours with a diges- 
tive fluid prepared by mixing- 1 part of pepsin-glycerine with 
1 part of pancreatin-glycerine, and 20 parts of a 0.3 per cent. 
solution of hydrochloric acid. Differences in the character of 
the protoplasmic cell-contents, and particularly in the dividing- 
nucleus, may be demonstrated by treating sections of fixed 
material with a digestive fluid made by mixing 1 part of 
pepsin-glycerine with 3 parts of water acidified with 0.2 per 
cent, of chemically pure hydrochloric acid. 

Diphenylamine. — This is a test for nitrates in plant tis- 
sues. Five centigrams of diphenylamine are dissolved in 10 
c.c. of pure sulphuric acid. The presence of nitrates is to be 
assumed when sections treated with this reagent take on a blue 
color. It seems, however, that in the presence of lignified 
tissues the reaction may fail, even when nitrates are present in 
abundance. Diphenylamine is also used to distinguish be- 
tween crystals of asparagin and potassium nitrate. Aspara- 
gin dissolves without color in this reagent, while potassium 
nitrate assumes a deep blue color on dissolving in it. 

Eau de Javelle. — Prepared by adding to an aqueous solu- 
tion of chloride of lime a solution of potassium oxalate so long 
as a precipitate is formed. The solution is then filtered and 
diluted somewhat with water before using. Or 20 parts of 
a 20 per cent, solution of calcium chloride is diluted with 100 
parts of water, and after this has stood for some time, a solu- 
tion of 15 parts of pure potassium carbonate in 100 parts of 
water is added. If a film should form on the surface of this 
on exposure to the air, a few drops of the solution of potas- 
sium carbonate should be added and the precipitate filtered 
away. 

Lignin is extracted from sections of woody tissues which 
have lain in the eau de Javelle solution for some time, and 



266 REAGENTS AND PROCESSES 

thereafter, on treating with chloroiodide of zinc, the mem- 
branes show only a cellulose reaction, staining only purple 
with the chloroiodide of zinc. 

Starch-grains included in chloroplasts may be demonstrated 
by first treating sections, or even whole leaves, with eau de 
Javelle until the chloroplasts are dissolved (this may take 
from one to twenty-four hours), and then treating the mate- 
rial with a solution of potassium iodide-iodine. The starch- 
grains will take on a blue or violet color. In some cases, 
however, the starch-grains themselves are dissolved with the 
eau de Javelle. In such cases, and indeed in most cases, 
chloral hydrate and iodine is to be preferred for demonstrat- 
ing starch-inclusions in chloroplasts (see under this head). 

When the forms of the cells simply are to be studied, eau 
de Javelle is very useful in clearing the sections by dissolving 
the cell-contents. If the sections become too clear in the eau 
de Javelle, this defect may be corrected by treating the sec- 
tions with alcohol or with a solution of alum. See under 
Cyanin for use of eau de Javelle in differentiating cutinized 
and suberized membranes. 

Eosin. — An aqueous solution of eosin is an excellent stain 
for protoplasmic cell-contents and cellulose walls. The solu- 
tion should be quite dilute. For the use of eosin in double 
staining see under Cyanin and Gram's Method. See also in 
the next chapter under Aleurone Grains. 

Fehling's Solution. — Prepare three separate solutions: 
(i) 17.5 gm. of copper sulphate in 500 c.c. of water; (2) 
86.5 gm. of sodium-potassium tartrate in 500 c.c. water; (3) 
60 gm. of caustic soda in 500 c.c. of water. To prepare for 
use, mix 1 volume of each of these with 2 volumes of water. 
The solutions keep well separately, but the mixture becomes 
changed after a time, and for this reason the solutions should 
not be mixed until needed. 

Sections may be treated with this solution on the glass slip. 
Two small drops of distilled water are placed on the slip with 
1 small drop of each of the three solutions ; then sections, not 



FERRIC CHLORIDE — FISCHER'S METHOD 267 

too thin, of the material which is to be tested for glucose are 
placed in the mixture on the slide. It is best to cut the sec- 
3 without wetting- the razor, and the sections should not 
be placed in water, but should be transferred directly to the 
mixture on the slide. The sections should be covered with a 
coverglass and the slide carefully heated over the flame of an 
alcohol lamp, or a very small flame from a Bunsen burner, 
until bubbles arise in the solution. If glucose is present, the 
sections will appear reddish from very small crystals of cu- 
prous oxide which have been reduced from the solution. If 
it is not desired to observe the crystals of cuprous oxide within 
the cells, but simply to demonstrate the presence of grape- 
sugar, small pieces of the tissues to be tested may be placed in 
a test-tube containing a few cubic centimeters of the solution, 
which is then heated to boiling; if grape-sugar is present in 
considerable quantity, a copious precipitate will after a time 
settle to the bottom of the tube. See under Copper Acetate. 
This is particularly suitable for demonstrating the presence of 
grape-sugar in those cells which contained it in the uninjured 
tissues. 

Ferric Chloride. — An aqueous solution is used as a test 
for tannin. When sections containing tannin are placed in 
this solution on the slide, a color is produced which may vary 
from dark blue to green. 

Ferricyanide of Potassium. — Used in demonstrating the 
structure of pyrenoids. Algse containing pyrenoids are placed 
in a mixture of equal parts of a 10 per cent, solution of ferri- 
cyanide of potassium and a 55 per cent, solution of acetic acid, 
and then treated as described under Dahlia. 

Fixatives and Fixation. — See page 226. 

Fischer's Method of Demonstrating Cilia. — The follow- 
ing method is highly recommended for demonstrating cilia of 
certain bacteria : An exceedingly small amount of the culture 
containing the bacteria is spread out as thinly as possible on 
the coverglass. After the film has dried on the coverglass 
the latter is passed through the flame of an alcohol lamp or 



268 REAGENTS AND PROCESSES 

Bunsen burner (care being taken to avoid a too excessive 
heat), and then a few drops of a mordant are put on the film 
on the coverglass. The mordant is prepared by dissolving 
2 gm. of tannin in 20 c.c. of water. The coverglass is then 
passed back and forth over a small flame until vapor arises 
from the mordant. The mordant is now washed off by means 
of water from a wash-bottle, and then one edge of the cover- 
glass is held in contact with a piece of filter paper to draw 
away the surplus water. Next, a concentrated aqueous solu- 
tion of fuchsin is spread over the film on the coverglass, and 
the coverglass is held over a flame until the fuchsin solution 
begins to boil; the coverglass is then washed off, and is al- 
lowed to dry. At any time thereafter the coverglass, with 
the film side down, may be cemented to the slide with balsam. 
In successful preparations made by this method cilia, when 
present, will stand out quite sharply. 

Fuchsin. — Dissolve 1 gram of fuchsin in 100 c.c. of abso- 
lute alcohol and 100 c.c. of water. An excellent single stain. 
Especially to be recommended for preparations that are to be 
photomicrographed. It stains different tissues different tints 
of red. 

An excellent double stain with fuchsin and methyl blue is 
obtained as follows. Leave sections in the above fuchsin solu- 
tion over night or for several hours. Wash the sections 
thoroughly in water and rinse in 95 per cent, alcohol quickly 
and remove them quickly to water while the stain is still com- 
ing off in clouds and transfer them to a saturated solution 
of methyl blue diluted with an equal bulk of water. Leave 
the sections in this for a few minutes, then rinse them in 
water and again in 95 per cent, alcohol; transfer them to 
xylene and mount them in Canada balsam. The time ratios 
for the two stains will vary with different materials, and the 
ratios are to be changed as experience teaches. 

Fuchsin, Acid. — Excellent for staining crystalloids. The 
material containing the crystalloids should be fixed in a con- 
centrated alcoholic solution of corrosive sublimate. Then the 



ACID FUCHSIN 269 

sections should be immersed Eor twenty four hours in a 0.2 

per cent, solution of acid fuclisin, to which a little camphor 
has been added. To demonstrate crystalloids in plastlds 
the sections should be treated as follows: The sections are 
placed in a solution of 20 per cent, acid fuchsih in 100 gm. 
o\ aniline-water. This solution is heated somewhat while 
the sections remain in it from two to five minutes; they are 
then rinsed in a solution of 1 part of a concentrated solution 
of picric acid in alcohol and 2 parts of water. This solution 
should be warmed to about 40 C, and the sections should be 
rinsed in it until they cease giving off color to it. Thereafter 
they are dehydrated in strong alcohol, passed into xylene, and 
mounted in Canada balsam. 

Acid fuclisin is an excellent stain for leucoplasts and plas- 
tids in general. The material is fixed in a concentrated alco- 
holic solution of corrosive sublimate in absolute alcohol, where 
the material remains for twenty-four hours ; then the fixa- 
tive is washed out in alcohol containing iodine. Sections 
from this material are placed in a 0.2 per ■ cent solution 
of acid fuchsin in distilled water. After remaining 
twenty- four hours they are taken out, washed in run- 
ning- water for a time, and are then examined in glycer- 
ine or are allowed to dry, after which they are mounted in 
Canada balsam. The sections cannot be dehydrated in alco- 
hol, because this will extract the stain from the plastids. 
The following method may also be used: The material is 
fixed in a solution of 5 gm. of corrosive sublimate in 100 
gm. of absolute alcohol, which is acidulated with 10 drops 
of hydrochloric acid. Then the fixative is removed by plac- 
ing the material in pure alcohol, which is several times re- 
placed. Sections from this material should be stained by im- 
mersion for about twenty minutes in a solution of 20 gm. 
of acid fuchsin in 200 c.c. of distilled water and 3 c.c. of 
aniline oil. They are then washed in a mixture of 50 c.c. of 
a saturated alcoholic solution of picric acid and 100 c.c. of 
water until color ceases to be given off from the sections. 



270 REAGENTS' AND PROCESSES 

Then the picric acid is washed from the sections in pure alco- 
hol. The sections are next placed in chloroform for ten min- 
utes and are then ready to be mounted in Canada balsam. 

When desired, sections cut from fresh material may be 
fixed and stained as above. Or the material may be fixed 
and imbedded, and after microtome sections have been cut 
and mounted on the slide they may be stained as above 
directed. 

A beautiful double stain for nuclei is prepared from acid 
fuchsin and methyl blue as follows: The microtome sections 
mounted on the slide are immersed for half an hour in a 0.00 1 
per cent, aqueous solution of acid fuchsin, then quickly washed 
in water, and immersed for about one minute in a 0.002 per 
cent, aqueous solution of methyl blue. The surplus stain is 
then washed off in alcohol and the preparation is allowed to 
dry; then the sections are immersed in olive oil from six to 
twenty-four hours, after which they are washed in absolute 
alcohol or in a mixture of absolute alcohol and xylol until the 
stains are quite clear, and the preparation is ready to be 
mounted in Canada balsam. 

Gelatine. — Motile swarm spores and the like are some- 
times mounted for observation in a solution of gelatine, which 
renders their movements less rapid, and in this way facilitates 
the study of these bodies. About 1 gm. of gelatine is dis- 
solved in 100 c.c. of water; a drop of this is placed upon a 
slide which has been somewhat warmed, and then a drop of 
the fluid containing the motile bodies is added to the drop of 
gelatine solution and mixed with it by stirring, after which 
the coverglass is put on. See also under Nutrient Media. 

Gentian Violet. — A 1 or 2 per cent, solution of acetic acid, 
to which gentian violet is added until the solution appears of 
a deep violet color, is effective in instantaneously fixing and 
staining the nuclei of fresh tissues. Anthers and sporangia 
need only to be teased out with a needle in this fluid or crushed 
under the coverglass, when the nuclei of the pollen-grains and 
spores or the mother cells of these will be fixed and stained 



GLYCERINE 2; I 

for immediate examination. See also page 23] and Gram's 
Method. 

Glycerine. — This is frequently used as a mounting medium, 
but since objects are apt to become very transparent in it, only 
those sections which have been stained should be mounted in 
it. Sections, such as of wood, which are not apt to shrink 
easily may be mounted in glycerine directly from water, but 
delicate tissues should first go from water into a mixture of 
10 parts of water and 1 part of glycerine; this should then be 
allowed to concentrate by the evaporation of the water, when 
the sections may be mounted on the slide in a drop of pure 
glycerine. The coverglass should be quite clean and the 
glycerine should not be allowed to run back over it. After 
putting on the coverglass the surplus glycerine should be 
taken up with a bit of filter paper and the slide about the 
edge of the coverglass .should be made quite clean with a cloth 
moistened in water and then wiped dry with a dry cloth ; then 
the slide may be put in position on the turntable, where a ring 
of Brunswick black, or of shellac to each ounce of which 20 
drops of castor oil have been added, may be spun around the 
edge of the coverglass. This process should be repeated sev- 
eral times, allowing each coat to harden before putting on the 
next, until a strong ring of the cement has been formed. 
When certain stains are used, such as hematoxylin, the glyc- 
erine must be entirely free from acids ; but with other stains, 
such as the carmine stains, an acidulation with 1 per cent, of 
acetic acid is of advantage. 

Dilute glycerine, in wdiich sufficient chrome-alum has been 
dissolved to give a clear blue color, is recommended as a 
mounting medium for the Schizophycese and Florideae, since 
the natural colors of these plants are retained in this medium. 

Sections containing mucilaginous membranes may be 
mounted in a drop of pure glycerine in which the membranes 
will not swell, and then, by irrigating the mount with water, 
the process of the slow swelling of the membrane may be 
observed. 



2J2 REAGENTS AND PROCESSES 

Glycerine-gelatine. — This is for most subjects a better 
mounting medium than glycerine alone. It is prepared as 
follows : One part by weight of the best gelatine is soaked for 
about 2 hours in 6 parts by weight of distilled water. Then 
7 parts by weight of chemically pure glycerine are added, and 
finally, to each ioo gm. of this mixture i gm. of concentrated 
carbolic acid. The mixture is warmed for about 15 minutes, 
and at the same time constantly stirred until it becomes clear ; 
then, by means of a hot-water funnel, or while kept warm in 
an incubator, the mixture is filtered through glass-wool or 
filter paper which has been washed with distilled water after 
being placed in the funnel. 

To mount sections in glycerine-gelatine the glass slip is 
warmed and a small bit of the gelatine is placed upon it. If 
the slip is not warm enough to melt the gelatine, it should be 
passed back and forth above the flame of an alcohol lamp. 
If the sections are of a character not liable to shrink, they may 
be transferred directly from water to the melted gelatine; if, 
however, there is danger of shrinking, the sections should first 
be placed in a 10 per cent, solution of glycerine, which is then 
allowed to concentrate by evaporation of the water, and then, 
from the concentrated glycerine the sections may be trans- 
ferred to the drop of melted glycerine-gelatine. To avoid 
air-bubbles the coverglass should be put on with the precau- 
tions given on page 232 for putting on the coverglass when 
Canada balsam is the mounting medium. If several sections 
are being mounted under one coverglass, and these should 
come to lie over each other in putting on the coverglass, they 
may be properly arranged without attempting to remove the 
coverglass (which usually makes the matter worse) by heat- 
ing the slide until the gelatine becomes quite soft, and then 
drawing a hair under the coverglass, with which the sections 
may be manipulated. It is sometimes a difficult matter to 
put just the right amount of the gelatine on the slip. To 
overcome this difficulty, heat the gelatine and pour it out in 
a thin film over a clean glass plate. When it has become cool, 



GLYCERINE GUM GRAM S METHOD 273 

strip it from the glass; then cut off small squares of different 
size, melt them separately on glass slips, and cover with the 
coverglasses of the size to be used with subsequent prepara- 
tions. The film of gelatine should then be cut into wafers of 
the si/e found to exactly fill out the space under the cover- 
glass. These wafers should be kept from drying too much 
and free from dust in tightly-stoppered bottles. 

Glycerine Gum. — Dissolve 10 grams of powdered gum 
arabic in 10 c.c. of water and add about 40 drops of glycer- 
ine. This is useful for imbedding hard seeds, pollen grains, 
etc.. preparatory to sectioning them. Put a drop of the glyc- 
erine gum on a suitable pine block and submerge the material 
in it, and leave the preparation in the air to dry. Sections 
may then be cut free-hand with a razor or on a microtome. 
Wash out the gum from the sections in water. 

Glycerine-iodine. — See under Iodine and Glycerine. 
Gram's Method. — This method is specially recommended 
for staining bacteria, either in coverglass preparations or in 
sections. The sections are stained in a mixture of 100 c.c. of 
aniline water (prepared by combining about 5 c.c. of aniline 
with 95 c.c. of distilled water), and 11 c.c. of a concentrated 
alcoholic solution of gentian violet, or, better, methyl violet. 
This is filtered, and 10 c.c. of absolute alcohol are added to it. 
The preparation is taken from the stain, rinsed in alcohol, and 
transferred to a solution of 2 parts of potassium iodide, and 
1 part of iodine in 300 parts of distilled water, where it re- 
mains from 1 to 3 minutes. Then it is rinsed in alcohol, trans- 
ferred to clove oil, and thence mounted in Canada balsam. A 
good double stain is obtained if the clove oil has some eosin 
dissolved in it. 

Gunther's modification of the Gram method is as follows : 
The preparation is stained and passed through the potassium 
iodide-iodine solution as above. Then it is placed for 1 to 2 
minutes in alcohol, next for 10 seconds in a 3 per cent, solu- 
tion of hydrochloric acid in alcohol, then again for several 
19 



274 REAGENTS AND PROCESSES 

minutes in pure alcohol, until no more color comes away, and 
then it is passed on into xylol, and finally is mounted in Can- 
ada balsam. 

Gum Arabic. — The study of the spermatozoids of ferns, 
etc., is facilitated by adding a 10 per cent, solution of gum 
arabic to the drop of water containing the spermatozoids, 
which are then unable to move so rapidly in the thicker fluid. 

Haematein. — Dissolve with heat i gm. of hsematein in 50 
c.c. of 90 per cent, alcohol, and add to this a solution of 50 
gm. of alum in 1 liter of distilled water. After cooling, filter 
if necessary, and add a crystal of thymol to prevent the growth 
of fungi. The solution is ready for use at once. Sections 
stained in this solution should be washed in water and trans- 
ferred to glycerine-gelatine for mounting, or may be dehy- 
drated and mounted in Canada balsam. The stain may be 
reduced in overstained sections by allowing the preparation to 
stand for some time in a 1 per cent, solution of alum. A 
sediment is apt to settle from this solution, but this is not an 
indication that the stain is spoiled. The sediment can be 
partly prevented by adding to the solution about 2 per cent, 
of glacial acetic acid, which, on the whole, increases the effec- 
tiveness of the stain. The acid should be entirely washed 
from the sections with water before permanent mounts are 
made. 

Haematoxylin, Delafield's. — Prepared by mixing 4 c.c. of 
a saturated solution of hematoxylin crystals in absolute alco- 
hol with 150 c.c. of a saturated solution of crystals of ammo- 
nium alum in water. After standing for a week exposed to 
the light and air, this should be filtered and mixed with 22 c.c. 
of glycerine and 25 c.c. of methyl alcohol. Before using this 
it should be allowed to stand until all precipitates have settled. 

Sections are transferred from water into the stain, where 
they remain for several minutes; they are then placed in a 
dish of 70 per cent, alcohol, acidulated with a drop of HC1, 
and after a minute they are rinsed in 75 per cent, alcohol, 
then in xylene, and mounted in balsam. 



HEMATOXYLIN— HARDENING PROCESSES 275 

Haematoxylin and Safranin. — Sections stained in safranin 

and washed in water may be placed for a few minutes in Dela- 

tield's haematoxylin, where they are treated as described under 

-tain. By this treatment lignified and snberized walls are 

stained red and cellulose walls violet. 

Hanging-drop Culture. — A hanging-drop culture is useful 
in the study of various microorganisms. Spin a ring of melted 
paraffin on the slide the size of the coverglass to be used. 
Wash the coverglass with soap and water, rinse it and rub 
it bright with alcohol on a clean cloth, and sterilize it by 
baking in an oven. Handle it thereafter with sterilized for- 
ceps. By means of a sterilized glass rod or pipette transfer 
a drop of nutrient solution (see under Nutrient Media) to the 
middle of the coverglass, and to this drop transfer with a 
sterilized needle a very minute portion of the culture or mate- 
rial to be studied. Invert the culture over the paraffin ring 
and press it down firmly so that a tight union is made. This 
is necessary to keep the drop from evaporating. If the 
paraffin is too hard to make a close union put a thin layer of 
vaseline over the ring before putting on the coverglass. The 
drop should of course hang free in the cell and not touch the 
slide. Sometimes it is desirable to draw the drop out a little 
over the coverglass with a sterilized needle, or even to flatten 
it out entirely by placing over it a coverglass enough smaller 
than the first not to touch the paraffin ring. 

The dealers furnish hollow-ground slides that are excellent 
for hanging-drop cultures. With these no paraffin ring is 
needed. 

Cultures prepared in this way can be studied at any time, 
even with high powers, without disturbing them. 

Hardening Processes. — The hardening of tissues is accom- 
plished by the withdrawal of water from them. This is in 
most cases, best accomplished by means of successively higher 
grades of alcohol, as described on page 227. 

A quick method of hardening fresh tissues, and at the same 
time preparing them for immediate sectioning, is to freeze 



276 REAGENTS AND PROCESSES 

them by the evaporation of ether or the expansion of liquid 
carbonic-acid gas. This process requires the use of special 
apparatus, for a description of which the student is referred 
to the catalogue of Bausch and Lomb, Rochester, N. Y. For 
an imbedding mass, either a drop of the white of egg, or a 
thick solution of dextrin in a solution of carbolic acid, 1 part, 
water 40 parts, may be placed about the object before freezing. 
If the dextrine solution is used, it would be better to pump the 
air from the object while immersed in the solution; then place 
the object on the object-holder, pour a small amount of the 
solution about it, and freeze. This method will answer very 
well in some cases, when it is desired to prepare a large num- 
ber of sections quickly for class use, but it can by no means 
take the place of fixing the material in an appropriate fixative, 
hardening slowly in alcohol, and imbedding in paraffin or 
collodion. 

The mucilaginous layer of certain seed coats may be har- 
dened with a 10 per cent, solution of neutral acetate of lead. 
The sections are cut from dry seeds, hardened in the lead 
acetate, and stained with methyl blue. They are then washed 
in water and mounted in a 2 per cent, solution of boracic acid. 

Hydrochloric Acid. — This reagent has such manifold appli- 
cation in histology that its uses are best learned in the specific 
cases of its application. See in the next chapter under Amy- 
lose, Berberin, Caffeine, Calcium Oxalate, Calcium Sulphate, 
Ethereal Oils, Magnesium Sulphate, Middle Lamella, Myro- 
sine, Pectic Substances, Phloroglucin, Theobromine, Vanillin. 
See also in this chapter under Maceration. 

Hydrogen Peroxide. — One part of hydrogen peroxide 
mixed with 20 parts of 60 per cent, alcohol will, in a few min- 
utes, remove from sections the dark discoloration due to osmic 
acid which has been used as a fixative (see page 226). 

India Ink. — The gelatinous sheath of the conjugate may 
be demonstrated by placing the alga under investigation in 
water in which India ink has been rubbed up until the water 



I N FILTRATION- [ODINE 277 

has a dark gray color. In this the gelatinous sheath becomes 
sharply demarked. 

Infiltration. — For infiltration with glycerine gum see page 
273, with paraffin, page 228, with collodion, page 233. The 
stony tissues of seeds, etc., which are too hard and brittle to 
be sectioned with a knife, and must, therefore, be. ground to 
the requisite thinness on a stone or by means of emery powder, 
may be protected against breaking during this process if fairly 
thin sections are first cut with a fine saw and then placed in 
a rather thin solution of Canada balsam or copal in chloro- 
form, which is then allowed to evaporate to the thickness of 
syrup; the sections are allowed to dry and are then cemented 
by means of a thick solution of gum arabic to a glass plate 
preparatory to grinding. Only a thin layer of gum arabic 
should be used, and this should be quite dry before the grind- 
ing is begun. The sections may now be ground thin on a 
clean, dry Arkansas or Wichita stone. Before the section has 
been brought to the desired thinness, the surface should be 
polished by rubbing it on a piece of soft leather which has 
been dressed with tripoli. The stone on wdiich the sections are 
ground may be cleaned of the balsam from time to time by 
means of a cloth dipped in xylol or turpentine. When one 
side has been polished, the section may be freed from the glass 
plate by soaking in w r ater, and then the polished side should 
be cemented to the glass plate and the reverse side ground and 
polished as before. The sections should be examined from 
time to time w^ith the microscope, so that the process of grind- 
ing may be stopped as soon as the desired transparency has 
been obtained. They may then be washed from the glass 
plate with water, and after drying should be mounted in Can- 
ada balsam. 

Iodine. — The fumes from heated crystals of iodine serve 
well in many cases as a fixative. Small objects in drop cul- 
tures may be readily fixed by pouring over them the fumes 
arising from iodine heated in a test-tube. Algae may be fixed 
by placing a few crystals of iodine in the bottom of a test- 



278 REAGENTS AND PROCESSES 

tube, cautiously inclining the tube slightly with the mouth 
downward, then placing the algae in the test-tube near the 
mouth directly from the water in which they were growing, 
and thereafter heating the crystals so that the fumes from 
them pour down over the algae. The iodine may afterward 
be expelled by warming the fixed material to 30 ° or 40 ° C, 
and the material will then need no further washing out. 

Iodine has a wide application in plant histology and micro- 
chemistry. See under Aconitine, Atropine, Carotin, Cellulose, 
Colchicine, Gums, Gram's Method, Lipochromes, Lignin, Nico- 
tine, Proteids, Suberin. 

Iodine and Alcohol. — A good fixative for very small or- 
ganisms is a solution of 3 parts of iodine in 100 parts of 70 
per cent, alcohol. This, at the same time, permits the staining 
effect of iodine on the cell-wall and cell-contents. 

Iodine and Glycerine. — A mixture of potassium iodide- 
iodine with glycerine in equal parts gives good results when 
the action of iodine is to be observed. The glycerine keeps 
the preparation from drying, and at the same time has a clear- 
ing effect. 

Iodine and Phosphoric Acid. — Used as a test for cellulose, 
which it colors violet. Prepared by dissolving with heat 0.5 
gm. of potassium iodide and a few crystals of iodine in 25 c.c. 
of concentrated aqueous solution of phosphoric acid. . 

Iodine and Potassium Iodide. — This solution is prepared 
by dissolving 0.5 gm. of potassium iodide and 1 gm. of iodine 
in a small amount of water, and then diluting this with 100 
c.c. of water. The solution is left standing over any iodine 
which may crystallize out. This formula is recommended by 
Arthur Meyer in his work on " Starkekorner " as best adapted 
to the study of starch-grains. A rough-and-ready method of 
preparing an iodine solution is to dissolve a small amount of 
potassium iodide in distilled water and then dissolve crystals 
of iodine in this until a brown color is obtained. This can be 
diluted with water as is found necessary. A rather pale solu- 



IODINE GREEN MACERATION 2/Q 

tion of iodine is sufficient to color starch blue. To stain modi- 
fied cell-walls the solution needs to be stronger. 

Iodine Green. — Sec page 233 for the use of iodine green in 
double staining. A 2 per cent, solution of glacial acetic acid 
with iodine green dissolved in it serves well in the instant 
fixing and staining of the nuclei of fresh material. 

Iron Acetate. — Used in the detection of tannins, which see 
in the next chapter. 

Lactic Acid. — Dried algae and fungi may be prepared for 
study with the microscope by soaking them first in water and 
then in concentrated lactic acid, in which they are heated until 
small bubbles are formed; they may then be studied in the 
lactic acid. A 10 per cent, solution of lactic acid is recom- 
mended for fixing bacteria. This fixative is said not to inter- 
fere in any way with the subsequent processes of staining with 
alcoholic solutions of aniline dyes. 

Lead Acetate. — A 10 per cent, solution of neutral lead 
acetate' is used to harden the mucilaginous layers of seed coats. 
For subsequent treatment see under Boracic Acid. 

Lithium Carbonate. — Useful in removing from material 
picric acid, which has been used as a fixative. A few drops 
of a cold, saturated, aqueous solution of lithium carbonate are 
added to the alcohol, which is used to wash out the fixative. 

Maceration. — In studying the forms of cells it is sometimes 
desirable and even necessary to isolate them by the process of 
maceration. Where cells with lignified walls are to be iso- 
lated Schultze's maceration process is best employed. Put a 
small amount of concentrated nitric acid into a test-tube and 
add a small crystal of potassium chlorate. Heat this to boil- 
ing and drop into it sections containing the tissues under inves- 
tigation. The sections will soon turn quite white. When 
this occurs, and before the sections have time to dissolve 
altogether, pour the contents of the test-tube into a large dish 
of water. Select a section and tease it out in a drop of water 
on a glass slide with dissecting needles, and examine the prepa- 
ration under a coverglass. Bast and wood fibers and stone 



280 REAGENTS AND PROCESSES 

cells can be isolated by this process, but thereafter they give 
the reaction for cellulose instead of for lignin. 

Another process known as the Mangin process gives good 
results with sections. Place the sections for forty-eight hours 
in a mixture of four volumes of alcohol and one volume of 
hydrochloric acid. Wash the sections in water and put them 
into a 10 per cent, ammonia for fifteen minutes, then mount a 
section in a drop of water under a coverglass and press upon 
the coverglass until the cells are forced apart. 

Chromic acid also is used for maceration. Place sections 
in a concentrated solution for a minute or so, then rinse them 
in water, mount them in a drop of water under a coverglass 
and press upon the coverglass. If the cells do not then come 
apart they should be put for a longer time in the acid. 

Methyl-blue. — An aqueous solution is an excellent stain 
for cellulose membranes. It may be used as a double stain 
with safranin as follows: Stain in safranin over night (see 
under Safranin) and then rinse in water and acidulated alco- 
hol; place in a concentrated aqueous solution of methyl-blue 
for fifteen minutes, rinse in strong alcohol and xylene and 
mount in balsam. See also under Fuchsin. 

Methylene-blue. — A good nuclear stain. For cells filled 
with protein granules it is particularly good in differentia- 
ting the nucleus. Methylene-blue is useful in differentiating 
pectin, compounds. The protoplast and lignified walls are 
stained a bright blue, while pectin compounds are stained a 
violet blue. Cells containing tannin will accumulate methylene- 
blue from very dilute solutions. The sections of living tissues 
are placed in a solution of i part of the stain in 500,000 parts 
of filtered rain-water. The cells containing tannin soon take 
on a distinct blue color, and, later, a deep blue precipitate is 
formed in them. The gelatinous sheaths of live conjugate 
may be stained by dilute aqueous solutions of methylene-blue 
without injury to the living organism. A 0.001 per cent. 
solution of methylene-blue in water will stain the living nuclei 
of diatoms and other simple organisms. The central body of 



M ETHYLENE BLUE -< s l 

the Cyanophyceae may be stained by the above dilute solution 
if, after twenty-four hours' treatment, the stain is strength- 
ened to a o.i per cent solution. Methylene-blue and carmine 
form a good differential stain for bacteria occurring in sections 
of tissues. 

Methylene-blue and Carbol-fuchsin. — This double stain- 
ing method is used in the differentiation of Bacillus tubercu- 
losis. The material first coughed up from the lungs by the 
patient im waking in the morning sbould be expectorated into 
a wide-mouthed bottle or co\*ered jar. The person who is to 
make the examination should afterward pour this out into a 
shallow glass dish. This should be placed on a dead-black 
background, and one of the small, yellowish, lenticular bodies 
which usually occur in tuberculous sputum should be removed 
and placed on a coverglass. A second coverglass should be 
placed over this ; then press the coverglasses gently between 
the thumb and forefinger, and rub to and fro until the material 
is spread out in a thin film on the coverglasses. Then slide 
the coverglasses apart, and allow them to dry in the open air. 
When dry, hold them with a pair of forceps and pass them 
three times through the flame of the Bunsen burner or alcohol 
lamp. (The film should not be allowed to turn brown, else 
the preparation will be ruined. ) Next pour over them carbol- 
fuchsin prepared by rubbing I gm. of fuchsin with ioo c.c. 
of a 5 per cent, aqueous solution of carbolic acid, with the 
gradual addition of 10 c.c. of alcohol. Hold the coverglasses 
over a flame with forceps until vapor begins to arise from the 
surface of the stain. Then hold away from the flame, except 
in intervals of gentle heating, by which they are kept warm 
for a minute or two. They are next washed in water and 
decolorized by being moved about in a 25 per cent, solution 
of nitric or sulphuric acid. When the previously deep-red 
color has changed to a greenish tint, the preparation is washed 
in 60 per cent, alcohol to remove the color set free by the acid. 
If any red color still remains, the preparation should be rinsed 
in water and again treated with the acid-bath. By the above 



282 REAGENTS AND PROCESSES 

process the fuchsin has been removed from everything but the 
tubercle bacilli. The double staining is accomplished by now 
pouring over the preparation a mixture of 3 parts of water 
with 1 part of a concentrated alcoholic solution of methylene- 
blue. After a few minutes the methylene-blue is washed off 
with water, and the preparation is allowed to dry; when dry, 
it may be mounted in Canada balsam. Other bacteria than 
the tubercle bacilli are decolorized by the acid-bath, and are 
subsequently stained blue by the methylene-blue. 

Methyl-violet. — Starch-grains may be stained by treatment 
with a dark aqueous solution of methyl- violet. If the starch- 
grains after staining are treated with a very dilute solution of 
calcium nitrate, the stain becomes deposited particularly in the 
less dense layers of the grains. Useful as a stain for elaio- 
plasts. See under this head in the next chapter. See also in 
this chapter under Staining Intra Vitam. 

Millon's Reagent. — This should be prepared fresh, as 
needed, by dissolving mercury in an equal weight of nitric 
acid and then diluting this solution with an equal weight of 
distilled water. Proteids are colored a brick-red with this 
reagent. Sections to be tested are to be mounted in a drop 
of the reagent on a glass slip. Warming the slip hastens the 
reaction. 

Nutrient Media. — Nutrient media must be sterilized by 
heat to keep them from spoiling and to make it possible to 
grow in them pure cultures — that is, cultures of organisms of 
any desired single species. Sterilization may be accomplished 
by steaming the medium for about twenty minutes each day 
on three days in succession, after having poured it into test- 
tubes or flasks which have previously been tightly plugged with 
cotton rolled into the form of a stopper of the proper size and 
baked in an oven until the cotton is slightly scorched. The 
tubes and cotton plugs should be baked together. Or, if an 
autoclav is available in which steam can be generated under 
pressure, and accordingly at a higher temperature than that 
of boiling water at ordinary atmospheric pressure, the cotton 



nutkii'n r mi: di \ 283 

plugs and tubes, or flasks, will not need to be baked, but may 
erilized, together with the nutrient medium already poured 
into them, by subjecting them for fifteen minutes to a tempera- 
ture of t 15 ('. in the autoclav. At this temperature a single 
sterilization suffices. 

A good artificial nutrient medium for yeasts is made by 
adding 0.05 per cent, of tartaric acid to a 10 per cent, solution 
of cane-sugar. A filtered aqueous extract of malted barley 
also gives good results.' To prepare this, barley is germinated 
until the plumule just begins to protrude; the barley is then 
dried and ground up, and water is poured over it until there 
is about twice as much water by volume as of the powdered 
malt. The water should stand over the malt, with occasional 
stirring, for about an hour, when it may be filtered off and 
sterilized. Sterilized grape juice is also an excellent nutrient 
medium for yeasts. . Cultures of yeasts grown in the above 
media may be made to produce spores in about twenty-four 
hours if some of the culture is tranferred to the surface of 
sterilized bits of flower-pot which are half submerged in water 
and kept covered by a bell- jar. 

Cohn's normal solution for the culture of bacteria is pre- 
pared as follows : Dissolve in 200 gm. of distilled water 1 gm. 
of acid potassium phosphate, 1 gm. of magnesium sulphate, 2 
gm. of neutral ammonium tartrate, and 0.1 gm. of calcium 
chloride. 

An infusion of meat for the culture of bacteria is prepared 
by covering finely chopped lean beef with water and allowing 
it to stand for twenty-four hours in an ice-chest, after which 
it is to be filtered through a muslin bag, using pressure of the 
hands to make the filtration more complete. The filtrate is 
then cooked and again filtered, and neutralized by the gradual 
addition of a solution of carbonate of soda. The solution 
should be tested with litmus paper, and the addition of carbo- 
nate of soda should cease as soon as neutralization is accom- 
plished. To this solution is added 0.5 per cent, of common 



284 REAGENTS AND PROCESSES 

salt. Ten gm. of peptone may be added to a liter of the 
infusion. 

In place of the meat infusion as prepared above, meat ex- 
tract may be used in the ratio of 4 to 5 gm. per liter of water. 

Bouillon is prepared by adding 1 liter of water to 1 pound 
of chopped lean beef. This is cooked for half an hour, then 
filtered and neutralized with carbonate of soda, then again 
boiled for an hour to precipitate albuminoids. After a final 
filtering the bouillon is poured into flasks or test-tubes and 
sterilized. 

Infusions of hay and dried fruits may also be used for 
nutrient media. A hay infusion for the growth of Bacillus 
subtilis may be prepared as follows : Chopped hay is placed 
in a beaker and barely covered with well water; this is kept 
in an incubator at a temperature of 36 C. for four hours, 
after which time the extract is poured off and diluted, if nec- 
essary, to a specific gravity of about 1.004. The extract is 
now poured into a flask which, having been closed with a 
cotton plug, is placed in a steam sterilizer and subjected to a 
gentle evolution of steam for about an hour. The flask is 
then placed in an incubator at 36 C. for a day or two, after 
which time a film produced by colonies of Bacillus subtilis will 
have formed over the surface of the extract. The spores of 
this bacterium are particularly resistant to heat, and for this 
reason while the spores of other bacteria are killed by the 
process of steaming, those of Bacillus subtilis still retain their 
vitality. 

Solid culture media may be prepared by adding to any of 
the fluid culture media a sufficient amount of a gelatinous 
substance to keep the mixture from liquefying at the tempera- 
ture of the laboratory, or, if desired, at the higher tempera- 
ture of an incubator. One of the most used of the solid 
media is prepared by adding to the peptonized infusion of 
meat, as above described, 10 per cent, of the best French gela- 
tine. The gelatine may be increased up to twice this amount, 
as the temperature may require. One hundred grams of gelatine 



X I I RUN r MEDIA 



28S 



is allowed to soak in i liter <>i the meat infusion until the gela- 
tine becomes swollen, and then a gentle heal is applied until 
the gelatine is completely dissolved. After the gelatine is dis- 
solved the solution should again be neutralized, if necessary, 
with carbonate of soda. When the solution stands at a tem- 
perature of about 50 C, an egg stirred up in 100 gin. of 
water is added while the mixture is stirred with a glass rod. 
The mixture is then kept at the boiling-point for about ten 
minutes. This coagulates the egg-albumin and clarifies the 
liquid. The clarified liquid is now* filtered by means of a hot- 
water funnel or while kept warm in an incubator, the high 
temperature being necessary for the reason that the mixture 
would become stiff at a low temperature, and so incapable of 
being filtered. The medium should be distributed while warm 
in sterilized test-tubes or flasks, which are then stoppered with 
baked cotton plugs. It should then be subjected to a tempera- 
ture of ioo° in the steam sterilizer for ten minutes at four 
successive intervals of twenty- four hours. For the reason 
that gelatine loses its power of solidifying at ordinary tem- 
peratures after being subjected to the temperature of boiling 
water for a long period, the time of each sterilization is nec- 
essarily reduced to about ten minutes and the number of steri- 
lizations is increased to four; whereas with other solidifying 
substances, such as agar-agar, the length of each sterilization 
may extend to one hour, and the number of sterilizations need 
be only two or three. 

In pouring the filtered medium into the test-tubes care should 
be taken not to get any of the medium on the upper portion 
of the tube where the cotton plug would be likely to come in 
contact with it, else the plug would later be difficult of removal. 

A solid nutrient medium which will remain solid at a higher 
temperature than the gelatine medium may be prepared from 
agar-agar, a substance obtained from certain gelatinous algae, 
as follows : Two gm. of the agar are broken into small pieces 
and soaked in cold water for twenty-four hours. Then the 
water is poured off and the swollen agar is added to 1 liter 



2S6 REAGENTS AND PROCESSES 

of the peptonized meat infusion. The mixture is boiled for 
several hours until the agar is completely dissolved. The solu- 
tion is then neutralized with a solution of carbonate of soda, 
filtered, distributed in flasks or test-tubes, and sterilized by 
steaming for i hour at two or three successive intervals of 
twenty- four hours. 

Cooked potatoes afford a solid nutrient medium which is 
quickly prepared and which is particularly adapted for the 
culture of chromogenic bacteria. Potatoes free from wounds 
are selected and scrubbed in water until they are perfectly 
clean, and the eyes and any unsound spots, if these could not 
be avoided, are cut out with a knife. Then the potatoes are 
placed for an hour in a solution of I part of mercuric chloride 
in 500 parts of water to disinfect the surface. They are next 
steamed for about an hour in a steam sterilizer, and after 
twenty-four hours the steaming is repeated for about half an 
hour. The sterilized potatoes are then placed in glass Petri 
dishes, are cut in halves with a sterilized table-knife, and the 
cut surfaces are inoculated. If the source of the inoculation 
is not a- pure culture, an isolation of forms may be approxi- 
mated by making long scratches over the surface of the potato 
with a sterilized platinum needle which has been in contact 
with the source of the inoculation. It will add to the security 
of the process of sterilization if each potato, before being 
placed in the bath of mercuric chloride, is wrapped in a piece 
of tissue paper, and so protected until it is cut open for 
inoculation. 

Another method of preparing potatoes which is, on the whole, 
more convenient and certain, is to cut out long cylindrical 
plugs from sound potatoes by means of a cork-borer or any 
metal tube of the proper size, and then to cut the potato cylin- 
ders very obliquely in two pieces, each of which is then to be 
placed in the bottom of a test-tube so that the oblique surface 
stands uppermost. After plugging the tubes with baked cot- 
ton, the potato cylinders are subjected to a temperature of 
ioo° C. in the steam sterilizer for one hour at three successive 



NUTRIENT MEDIA 



87 



intervals of twenty-four hours. A sterilized paste made from 
potatoes or bread serves well for the culture of molds as well 
as of bacteria. 

A decoction of horse-dung- furnishes a good medium for the 
culture of Mucor and various other molds. The decoction is 
prepared by boiling the dung in water, then filtering and ster- 
ilizing the solution. By placing the dung of different kinds 
of animals in a moist chamber, as, for instance, in dishes 
floating on water and covered with a bell-jar, characteristic 
fungi will after a time appear on it. 

Single spore cultures of Mucor may be obtained in the fol- 
lowing manner : Glass slides are thoroughly cleaned and steri- 
lized by baking. By means of sterilized forceps a single spo- 
rangium of Mucor is picked from a spontaneous growth of 
this fungus on horse-dung or stale bread kept in a moist 
chamber. The sporangium is placed in a sterilized decoction 
of horse-dung contained in a sterilized watch-glass, which 
may be placed on an inverted tumbler in a plate of water 
and then covered with a bell-jar which should dip into the 
water and form a germ-proof moist chamber. After a few 
hours the sporangium will have burst open and the spores, 
which are now distributed through the decoction, will have 
swollen to several times their original diameter, and can all 
the more readily be discerned in subsequent manipulations. 
A needle which has been disinfected by heating in a flame is 
now dipped into the decoction and the point of it drawn along 
the surface of a glass slide which has been cleaned and steri- 
lized as above directed. By this process the decoction which 
has adhered to the needle is drawn out in the form of a nar- 
row streak, and if several spores of Mucor are present, they 
will be separated from each other. A single spore may be 
located with a medium power of the compound microscope, 
and all other spores present in the streak may be wiped off 
with a cloth which has been sterilized by heat. Then a drop 
of the decoction of sterilized horse-dung should be added to 
the small amount containing the spore on the slide. The slide 



2%% REAGENTS AND PROCESSES 

should be placed in a moist chamber where the spore will soon 
give rise to a mycelium visible to the naked eye, and from the 
mycelium numerous sporangia will be produced after a time. 
The slide may be taken from the moist chamber from time to 
time and the stages in the development of the fungus exam- 
ined, but as much care as possible should be taken to prevent 
the contamination of the culture. 

Knop's nutrient solution, which is particularly good for the 
culture of algae, consists of 4 parts of calcium nitrate, 1 part 
of magnesium sulphate, 1 part of potassium nitrate, 1 part 
of potassium phosphate. These should be dissolved in suffi- 
cient water to make a 0.2 per cent, or 5 per cent, solution of 
the combined salts. The potassium salts should first be dis- 
solved, then the magnesium salt, and last the salt of calcium 
should be added after having been dissolved by itself. By this 
procedure only a small amount of insoluble calcium phosphate 
is formed. The zoospores of Vaucheria may be induced to 
form at almost any time by transferring this alga from the 
above solution, in which it has been growing exposed to a 
bright light, to pure water; or cultures in a 0.1 per cent, or 
0.2 per cent, nutrient solution which have been exposed to the 
light need only be placed in a dark place in order to incite the 
production of zoospores. 

A 2 per cent, to 4 per cent, solution of cane-sugar may be 
used as a nutrient medium for algse. Filaments of Spirogyra 
may be made to conjugate by transferring them from the 
water in which they have been growing to a solution of cane- 
sugar as above, which is then placed in a well-lighted place. 

The formation of zoospores may be incited in CEdogomum 
by transferring filaments of the alga from water at a low tem- 
perature (say at the temperature of the early morning) to a 
2 per cent, or 3 per cent, solution of cane-sugar which is kept 
at a constant temperature of about 26 ° C. 

Convenient flasks for the preservation of sterilized fluid 
nutrient media may be made from glass tubing as follows : A 
piece of glass tubing 0.2 inch in diameter, or larger, is held 



NUTRIENT MEDIA - >s <) 

with its lower end in the flame of a blow-pipe, the tube being 
constantly revolved about its long axis to insure an even heat- 
ing of the end of the tube until the end of the tube becomes 
soft and just begins to draw downward in the form of a large 
drop. By this time the mouth of the tube has become closed. 
Then quickly the tube is removed from the flame, and while 
the melted end of the tube is still held downward, air is blown 
in at the upper end of the tube by means of the mouth, so that 
the molten glass at the lower end of the tube is forced out- 
ward in the form of a rounded flask. After cooling so that 
it may be handled, the tube is held in the flame close to the 
bulb, and by constant turning the tube is heated equally on all 
sides until it becomes so soft that it may be drawn out. This 
process is accomplished by taking the tube from the flame and 
pulling on it gently so that it may be drawn out quite long and 
narrow. The length of the stem of the bulb should be equal 
to the depth of the vessel from which the nutrient medium is 
to be drawn into the bulb. The stem may be severed from the 
tube by holding it in the flame of the blow-pipe at the proper 
distance from the bulb, where it will soon become soft enough 
to be pulled off from the main tube. Then the end of the 
capillary neck is held in the flame until a bead is formed; in 
this way the flask is hermetically sealed. To fill the flask with 
nutrient fluid the neck is sterilized near the end by passing it 
through a flame, and the head is broken off with sterilized 
forceps. The bulb is then heated in the flame of an alcohol 
lamp or Bunsen burner to expand the air. The end of the neck 
is next quickly dipped into the nutrient fluid, which is forced 
up the neck into the bulb as the air in this cools. When the 
bulb is two thirds full, the neck is withdrawn from the fluid 
and hermetically sealed in a flame. In filling the bulb the 
greatest care must be taken to keep the stock of nutrient me- 
dium from any source of contamination, if it has once been 
sterilized. Chemical flasks with narrow necks serve well for 
a common receptacle. These should be kept stoppered with 
20 



29O REAGENTS AND PROCESSES 

a cotton plug, and to fill the small flasks the plugs need only 
to be lifted slightly while the sterilized capillary neck of the 
small flasks is thrust past the plug into the nutrient fluid. If 
the nutrient fluid is freshly prepared, and has not yet been 
sterilized, the small flask may be filled, sealed up in the flame, 
and sterilized in the steam sterilizer or in a vessel of boiling 
water for an hour each day on three successive days. The 
nutrient fluid will keep indefinitely in the little flasks, and 
when a drop is wanted for a drop culture, it is only necessary 
to sterilize the end of the capillary neck in a flame, break off 
the bead with sterilized forceps, invert the flask, and place the 
palm of the hand over the bulb. The heat of the hand will 
expand the air over the fluid and force the latter down the 
neck. With a little practice just the desired amount of fluid 
can be forced out by the heat of the hand. The hand must 
not be placed on the bulb until the flask is inverted. If it is 
desired to make cultures within the little flasks, snip off the 
end of the capillary neck as before, and thrust a long platinum 
needle, the end of which has been in contact with the source 
of inoculation, down the neck into the fluid. Then withdraw 
the needle and hermetically seal the neck in the flame. When 
cultures are to be made in the flasks, these should be only one 
third filled by the nutrient medium; there will then be suffi- 
cient air in the flasks for the success of the culture after the 
flasks have heen inoculated and hermetically sealed. 

Pollen grains may be made to germinate in hanging-drops 
composed of 100 parts of well-water, 3 to 30 parts of cane- 
sugar, and 1.5 parts of gelatine. This should be made as 
needed, or it may be sterilized and kept indefinitely in the little 
flasks just described. The amount of cane-sugar to give the 
best results varies with the species of pollen, and can only be 
determined by experiment, but 3 parts will probably answer 
for most pollen grains. 

Spores of ferns may be made to germinate on pieces of 
flower-pot which are kept half submerged in water and are 
covered by a bell- jar. They should be set before a north win- 



OSMIC ACID PEPSIN 2QI 

dow. They should never be exposed to the direct light of the 

sun, since in such a position the temperature under the bell-jar 
would become very great. 

Osmic Acid. — The method of preparing- a solution of osmic 
acid and of its use in Flemming's fixative is given on page 226. 
The vapor of osmic acid may be used as a fixative for very 
small organisms. In order to accomplisb this a drop of water 
containing the organisms need only to be inverted over a bottle 
containing a 2 per cent, solution of the acid. Osmic acid 
colors ethereal and fatty oils from brown to black, but other 
organic substances are also darkened by it; and as a test for 
oils it is not absolutely reliable. Aleurone grains in sections 
of Ricinus which have been freed from their oil by standing 
for a time in strong alcohol may be stained brown, and the 
crystalloid and ground substance differentiated by immersing 
the sections for a short time in a 1 per cent, solution of osmic 
acid. 

Paraffin. — The directions for imbedding material in paraffin 
are given on page 228. Paraffin of about 52 ° C. melting point 
sections to good advantage at a temperature between 21 ° and 
24 C, or 70 ° and 75 ° F. Good cells for hanging-drop cul- 
tures may be made by placing glass slides on the turn-table 
and spinning rings on them by means of a earner s-hair brush 
dipped in melted paraffin. 

Pepsin. — One part of pepsin-glycerine and 3 parts of water 
acidulated with 0.2 per cent, of chemically pure hydrochloric 
acid. When sections containing protoplasts are subjected to 
this reagent at blood temperature, certain structures of the 
protoplast which are insoluble in the reagent may be isolated 
from those which are soluble. In the dividing nucleus the 
kinoplasmic spindle-fibers persist after the chromosomes and 
nuclear plate have been dissolved by this reagent. By the 
action of digestive ferments on aleurone grains the ground 
substance is first dissolved and then the crystalloid more 
slowly, while the limiting membrane of the vacuole occupied 
by the aleurone grain persists. Digestive ferments are thus 



2 9 2 REAGENTS AND PROCESSES 

found to be excellent reagents for demonstrating the differ- 
ence in constitution of the finer structures of the protoplast 
and protoplasmic cell-contents. 

Phloroglucin. — This furnishes one of the most reliable tests 
for lignin. Sections are placed in alcohol containing a trace 
of phloroglucin, transferred to a drop of water on a slide and 
covered with a coverglass. A drop of hydrochloric acid is 
then applied to the edge of the coverglass and, as the acid 
comes in contact with the lignified membranes, these are col- 
ored a bright violet-red. 

Phospho-molybdic Acid. — This is used as a test for pro- 
teids. Sections are treated for an hour or two with a solution 
of i gm. of sodium-molybdenum phosphate in 90 gm. of dis- 
tilled water and 5 gm. of concentrated nitric acid. Proteid 
materials then take on the appearance of yellow granules. 

Picric Acid. — The structures of aleurone grains are well 
differentiated by fixing in a concentrated alcoholic solution of 
picric acid and subsequent staining with eosin. The sections 
are to remain in the alcoholic fixative for several hours. They 
are then to be washed out in alcohol and stained for a few 
minutes in a solution of eosin in absolute alcohol. Then the 
sections are successively washed in absolute alcohol, trans- 
ferred to oil of cloves, and mounted in Canada balsam. The 
ground substance is dark red, the crystalloid yellow," while 
the globoid remains colorless. The pyrenoids and chloro- 
plasts of algae may be simultaneously fixed and stained by 
placing the algae for an hour or longer in a concentrated solu- 
tion of picric acid in 50 per cent, alcohol, to which has been 
added about 5 drops of a solution of 20 gm. of acid fuchsin 
in 100 c.c. of aniline water. The aniline water is prepared by 
shaking up 3.5 gm. of aniline in 96.5 gm. of water. The algae 
are then washed in alcohol, transferred to xylol, then to a thin 
solution of balsam in xylol, and are finally mounted in the 
thicker solution of Canada balsam in xylol. 

Alcohol is a better solvent of picric acid than water, and 
accordingly it gives quicker results in washing out the acid 



PICRO-ANILINE BLUE — POTASSIUM HYDRATE 293 

from the fixed material than water does, but running water 
may be used to wash out the fixative whether the latter has 
been dissolved in alcohol or in water. 

Picro-aniline Blue. — A double stain, which is very rapid 
in its action, is prepared by adding aniline blue to a saturated 
solution of picric acid in 50 per cent, alcohol until the solution 
has a blue-green color. By this treatment the unmodified cell- 
walls and the cell-contents are stained blue, while the lignified 
walls are stained by the picric acid. 

Picro-nigrosin. — A solution of nigrosin in a concentrated 
solution of picric acid in water or 50 per cent, or 95 per cent, 
alcohol is a good fixative and stain for algae and leucoplasts, 
and for double-staining modified and unmodified cell-walls. 
The solution may, in some cases, need to act for twenty-four 
hours. The strong alcoholic solution is particularly recom- 
mended for material containing chlorophyll, since this will be 
extracted by the strong alcohol. Nuclei and leucoplasts are 
stained a steel blue by the nigrosin. 

Potassium Alcohol. — Used for bleaching sections. It may 
be prepared by mixing a concentrated aqueous solution of 
potassium hydrate with 90 per cent, alcohol until a sediment 
is formed. This is allowed to stand for twenty-four hours 
with frequent violent shaking, and then the clear liquid is 
poured off and is diluted for use with 2 or 3 parts of water. 

Potassium Hydrate. — For general use, dissolve 5 gm. of 
potassium hydrate in 95 c.c. of distilled water. This is used 
as a clearing agent for sections and small organisms. The 
process of clearing may require from several hours to several 
days. After clearing, the potash should be washed out in 
plenty of water, and then the preparation may be neutralized 
with acetic acid. This will tend to make the objects more 
opaque, and if too much is added, the objects may be cleared 
again by caustic potash or ammonia. A dilute solution of 
caustic potash, as above, may be used for the maceration of 
cork, while delicate tissues in general may be macerated by 
boiling for a few minutes in a 50 per cent, solution of potas- 



294 REAGENTS AND PROCESSES 

sium hydrate in water; the tissues should then be washed in 
water and teased out on a slide in a drop of water. 

Ruthenium Red. — An aqueous solution is an excellent stain 
for pectic substances and for gums and slimes which have been 
derived from these. Ruthenium red is not soluble in alcohol, 
clove oil, or glycerine, and, therefore, preparations stained by 
it may be dehydrated and mounted in glycerine or balsam. 

Safranin. — A saturated solution of safranin in alcohol 
should be made and this should be diluted with an equal bulk 
of water, or with an equal bulk of a saturated aqueous solu- 
tion of safranin. This is an excellent general stain, and gives 
good differentiating effects when used singly. It is one of 
the few stains which are particularly adapted to the staining of 
pectic compounds. It also gives beautiful results in staining 
the cell-contents of Spirogyra and other algae. The algae, 
after fixing in a fixative containing chromic acid, should lie 
in the alcoholic solution diluted with an equal bulk of water 
for twelve or twenty- four hours. They should be transferred 
to 50 per cent, alcohol, to which strong alcohol is then added, 
drop by drop. The color will begin to be extracted in the 
alcohol, and when the right intensity has been reached, the 
material should be transferred to dilute glycerine, where it 
is to remain while the glycerine slowly concentrates in a place 
protected against dust. Then permanent mounts may be made 
in glycerine or glycerine-jelly. The stain given by safranin 
is quite permanent. See also page 231, and the directions there 
given for the three-color method. 

Salicylate of Soda. — A clearing reagent which for small 
objects is not inferior to chloral hydrate is furnished by dis- 
solving crystals of salicylate of soda in an equal weight of 
distilled water. With tincture of iodine added this reagent 
will cause starch to swell, at the same time imparting a blue 
color to it. 

Salt. — A 4 per cent, or stronger solution of common salt, 
or of potassium nitrate, may be used to cause plasmolysis in 



SHELLAC STAINING INTRA VITAM 



295 



living cells. This process may be all the more clearly seen by 
adding eosin to the salt solution. 

Shellac. — A thick solution of shellac in alcohol, to each 
ounce of which are added 20 drops of castor oil, makes an 
excellent sealing medium for preparations mounted in glycer- 
ine or glycerine-jelly, or in an aqueous medium. 

Silver Nitrate. — A solution of silver nitrate is used to bring 
out more clearly the striations in bast fibers and starch-grains. 
Sections containing striated bast fibers are allowed to dry and 
are then impregnated with the silver salt. Without previous 
washing the sections are transferred to a 0.75 per cent, solu- 
tion of common salt. They are then placed in distilled water 
and exposed to the light for a considerable time; thereafter 
they are allowed to dry and may be examined to good advan- 
tage in anise oil. 

Dry starch-grains are put to soak in a 5 per cent, solution 
of silver nitrate. After a time they are allowed to dry super- 
ficially and are then treated with a 0.75 per cent, solution of 
common salt, in which they are finally exposed to the direct 
light of the sun to reduce the chloride of silver which has been 
formed within the grains. The less dense laminae of the 
starch-grains will show a gray color, due to the reduced silver. 
See page 179 for a description of the structure of starch-grains. 

Staining Intra Vitam. — Living protoplasts may accumu- 
late certain stains from very dilute solutions without injury 
to themselves. Dahlia, methyl-violet, mauvein, and methylene- 
blue are particularly suitable for this purpose. Solutions 
containing 0.001 per cent, or 0.002 per cent, of any of the 
first three stains have given good results in staining living 
nuclei, while 1 part of methylene-blue in 500,000 parts of 
filtered rain-water is used for staining living cells containing 
tannin. A large amount of these very dilute solutions should 
be employed in order that a sufficient amount of coloring mat- 
ter may be at hand for accumulation by the living cells. Liv- 
ing protoplasts have the power of reducing and accumulating 
metallic silver from solutions of certain of the salts of silver, 



296 REAGENTS AND PROCESSES 

while dead protoplasts have not this power. The simplest 
method of producing this reaction is to place a few filaments 
of Spirogyra in a liter of a mixture of 1 part of silver nitrate 
in 100,000 parts of water with 5 c.c. of lime water. The 
experiment will be completed in about half an hour if the 
temperature is approximately 30 ° C. 

Tannin and Antimonium-potassium Tartrate. — These 
are used successively as a mordant for methyl- and gentian- 
violet, fuchsin, and safranin when sections stained with these 
are to be mounted in glycerine. The sections before staining 
are placed in a 20 per cent, solution of powdered tannin in 
cold water. After washing well in distilled water, they are 
placed for 24 hours in a 2 per cent, solution of antimonium- 
potassium tartrate. After washing again in distilled water, 
they are transferred to the stain. From the stain the sections 
are washed quickly in distilled water and placed in strong 
alcohol, where the color is washed out until the desired degree 
of intensity is reached. They are now ready for mounting in 
glycerine, or, if desired, they may be placed in xylol and then 
mounted in balsam. If the sections are so deeply stained that 
they cannot be sufficiently washed out in alcohol, they should 
be placed for a time in a 2.5 per cent, solution of tannin. 

Turpentine. — This may be used to dissolve paraffin from 
sections which have been cut from material imbedded in paraf- 
fin. See also under Carbolic Acid. 

Venetian Turpentine. — To prepare a mounting medium 
from Venetian turpentine the product as it comes from the 
apothecary is diluted with an equal volume of strong alcohol, 
and after the mixture has become clear by long standing or 
by filtering after being well shaken, it is thickened somewhat 
on the water-bath. Objects may be mounted directly from 
strong alcohol into Venetian turpentine as above prepared. 
Objects which are found to shrink by this treatment may be 
transferred from strong alcohol to a mixture of 10 parts of 
the turpentine with 100 parts of alcohol. The alcohol is then 
to be withdrawn from this mixture by placing the latter, to- 



XYLENE 



297 



gether with a dish of calcium chloride, under a bell-jar. In 
order to keep the mixture of turpentine and alcohol from 
mounting the sides of the vessel which contains it, the rim 
of the vessel should be coated over with hot paraffin. The 
turpentine hardens quite slowly, and in order to quickly fasten 
a c< 1 to the slide when the turpentine is being used 

for a permanent mount, a wire which has been heated in a 
flame should be quickly drawn around the edge of the cover- 
glass. 

Xylene. — This is used as a solvent for paraffin, either in 
removing paraffin from sections or in preparing a dilute solu- 
tion of paraffin to be used in the gradual infiltration of tis- 
sues with this substance. Used also as a solvent of Canada 
balsam. Xylol is the trade name for xylene. 



CHAPTER XVI 
MICROCHEMISTRY OF PLANT PRODUCTS 

Aconitine, C 33 H 45 N0 12 . — An alkaloid occurring in es- 
pecial abundance in the rootstocks of Aconitum Napellus. To 
demonstrate aconitine treat sections with potassium iodide- 
iodine, or with a solution of potassium permanganate. The 
first reagent produces a carmine-red coloration in the presence 
of aconitine and the second gives a red precipitate of aconitin 
permanganate. 

Aleurone. — See page 185 for a description of the nature 
of aleurone grains. The protein nature of aleurone is dem- 
onstrated by its dissolving with a red color in Millon's reagent 
and by its being colored yellow or brown with iodine reagents. 
Aleurone grains should be studied in a mixture of equal parts 
of castor oil and 95 per cent, alcohol slightly colored with eosin. 
In water they are in danger of going more or less into solu- 
tion. Permanent preparations of the aleurone of Ricinus may 
be made by placing small bits of the endosperm in a saturated 
alcoholic solution of picric acid, rinsing in alcohol, imbedding 
in paraffin (see page 228), sectioning on the microtome (see 
page 229), staining in an alcoholic solution of eosin, rinsing 
in oil of cloves, and then in xylene, and mounting in balsam. 
By this process the ground substance should be red, the crys- 
talloid yellow, and the globoids colorless. For reaction of 
aleurone to other reagents see in the last chapter under Borax- 
Carmine, Digestive Fluids, Pepsin. 

Alkaloids. — Sections to be tested for alkaloids should be 
thick enough to leave one cell layer intact. In order to make 
the determination of the alkaloid more certain, sections for 
control should be soaked for a day or so in a solvent of alka- 
loids prepared by dissolving 1 part of tartaric acid in 20 parts 

298 



AULYL SII.I'II IDK AMYI.ODKXTRINE 



•99 



of alcohol, and rinsing in water for a day to wash out the 
acid. Mount sections thus treated under a coverglass with 
untreated sections and apply reagents for detecting- alkaloids. 
The following reagents give with alkaloids amorphous or 
crystalline precipitates: potassium iodide-iodine, potassium 
bismuthiodide, chloroiodide of zinc, potassium-mercuriciodide, 
chloride of gold, ammonium-molybdate, potassium perman- 
ganate. See under Aconitine, Atropine, Berberin, Brucine. 
Caffeine, Corydalin, Curarin, Cytisin, Morphine, Narceine, 
Narcotine, Nicotine, Piperine, Sinapine, Strychnine, Theo- 
bromine, Veratrine. 

Allyl Sulphide or Garlic Oil, (C 3 H 5 ) 2 S. — This may be 
demonstrated by treating sections of species of Allium with 
palladous nitrate which produces a kermes-brown precipitate ; 
or sections may be treated with a solution of silver nitrate, 
when sulphide of silver will be formed. 

Amygdalin, QoE^NOn. — This nitrogenous glucoside is 
particularly abundant in bitter almonds and in the bark, leaves, 
and flowers of Prunus padus. It can be extracted in boiling 
water, and on addition of alcohol it crystallizes out in the 
form of pearly scales. It is split into prussic acid, oil of 
bitter almonds and sugar by the enzyme emulsin which occurs 
associated with the glucoside. 

Amylodextrine. — This carbohydrate occurs in those starch- 
grains which take on a reddish color with iodine, and it is 
formed by the action of diastase and acids from the amylose 
of those starch-grains which are colored blue with iodine. By 
the action of diastase on the starch of germinating seeds the 
amylose of the starch is converted first into amylodextrine, 
and this in turn into dextrine and isomaltose. The micro- 
chemical behavior of amylodextrine is given by Arthur Meyer 
as follows : Water at 70 ° C. dissolves crystals of amylodex- 
trine slowly, while at ioo° the crystals are dissolved at once. 
A solution of 10 gm. of pure calcium nitrate in 14 gm. of water 
dissolves crystals under the coverglass very slowly. After 



300 MICROCHEMISTRY OF PLANT PRODUCTS 

some hours, if a solution of iodine is added, the calcium ni- 
trate solution is colored brown, which indicates that the crys- 
tals of amylodextrine have at least been partially dissolved. 
A solution of 2 gm. of purest potassium hydrate in 100 gm. 
of water dissolves small crystals within 2 hours, while the 
solution of larger crystals requires a longer time. A solution 
of iodine, prepared as directed on page 277, colors the crystals 
dark brown. A 25 per cent, solution of hydrochloric acid 
dissolves large and small crystals immediately. When this 
solution is diluted with 4 parts of water, it takes on a brown- 
ish-red color with the iodine solution. When 1 drop of malt 
extract is added to 5 drops of a neutral solution of amylo- 
dextrine this becomes inverted within 10 minutes, so that it 
no longer is colored by the iodine solution. To prepare the 
malt extract treat 1 part of malt with 3 parts of water and 
filter the solution. The solution of crystals of amylodextrine 
by the malt extract requires several days. At a temperature 
of 40 ° C. saliva dissolves the amylodextrine crystals within 
48 hours. To prepare the saliva mix human saliva with a 
drop of chloroform, filter, and preserve over a few drops of 
chloroform. 

Amyloid. — This carbohydrate occurs as reserve material in 
the seeds of Tropceolum majus, Impatiens balsamina, Pceonia 
officinalis, and in many other plants. It is colored blue by 
dilute solution of iodine, but with a concentrated solution it 
is colored a brownish-orange. It is soluble in cuprammonia 
only after a day. Treated with a 30 per cent, solution of 
nitric acid it swells strongly, and finally dissolves. This is 
different from the amyloid produced by the action of acids and 
certain chlorides on cellulose. 

Amylose. — Starch-grains which are colored blue by iodine 
— that is, most starch-grains — are, according to Meyer, com- 
posed of crystals of two kinds of amylose, named by Meyer 
a-amylose and ^-amylose. The a-amylose has been isolated 
in crystalline form, but the ^-amylose has not been iso- 



ANH ' 



30 



lated, and its microchemical behavior has only been deter- 
mined by experiments with starch-grains. The microchem- 
ical behavior of the o-amylose is as follows, the reagents 
being prepared as directed under amylodextrine : Water at 
from 60 to too° C. does not soon dissolve the crystals of this 
amylose. Treatment with the calcium nitrate solution for 30 
minutes does not appear to affect the crystals. The solution 
of iodine does not color the crystals at first, but after a longer 
time it imparts a brownish color. The solution of hydro- 
chloric acid dissolves the crystals at once, and the solution, 
diluted with four times its bulk of water, is colored deep blue 
with the iodine reagent; but after the solution has stood for 
12 hours it is colored brownish or not at all by the iodine. 
The solution of potassium hydrate at ordinary temperatures 
affects the crystals so that they are colored blue by the iodine 
after the solution has been neutralized with acetic acid. In 
boiling potassium hydrate the crystals are changed into viscid 
drops. If the solution is now neutralized with acetic acid and 
diluted with four times its bulk of water, it takes on a deep 
blue color with the iodine reagent. 

If a drop of malt extract is added to the solution formed by 
boiling crystals of a-amylose with the potassium hydrate solu- 
tion, and exactly neutralizing with acetic acid, it is found after 
5 minutes that the solution takes on a red color, due to the 
formation of amylodextrine by the influence of the malt ex- 
tract. Saliva and malt extract have very little effect upon 
a-amylose. After treatment with these reagents for 15 days 
at a constant temperature of 40 ° C, no essential change could 
be detected. 

P- Amylose is insoluble in cold water, but at a temperature 
of 70 C. it forms viscid masses or minute droplets. The 
solutions of calcium nitrate, potassium hydrate, and hydro- 
chloric acid have the same effect as water, excepting that the 
solution in hydrochloric acid is more complete than in water. 
The solution of /3-amylose acts precisely as the solution of 



302 MICROCHEMISTRY OF PLANT PRODUCTS 

a-amylose. Undissolved /?-amylose, however, is colored blue 
by the iodine solution. The swelling of starch in hot water 
is probably due to the /?-amylose which it contains. Meyer 
considers a-amylose and /3-amylose to be the same substance, 
but that the latter contains water of crystallization, while the 
former does not. 

Anthochlorin. — A yellow coloring matter occurring in 
solution in the cell sap and differing from the yellow coloring 
matter xanthin occurring in chromoplasts in that it is not 
changed to a blue color by the action of concentrated sulphuric 
acid. 

Anthocyanins. — These are coloring matters of flowers, 
leaves, and other parts of plants which impart red, violet, blue, 
blue-green, or green colors, the character of the color being 
dependent on the alkalinity or acidity of the cell-sap. The 
anthocyanins are soluble in water, alcohol, and ether, and are 
decolorized in strong alkalies. 

Anthoxanthin. — This yellow coloring matter in the chro- 
moplasts of flowers and fruits takes on a blue color with con- 
centrated sulphuric acid. Since the chromoplasts of flowers 
and fruits were first of all green, anthoxanthin is probably a 
derivative of chlorophyll. Anthoxanthin is also called xan- 
thin and xanthophyll. 

Arabin. — This is the gum derived from species of Acacia 
and known as gum arabic. Arabin is soluble in hot and cold 
water, and insoluble in alcohol and ether. The aqueQus solu J 
tion will mix with glycerine, but concentrated glycerine has 
little effect on the hard gum. 

Asparagin, C 2 H 3 NH 2 .CONH 2 .COOH.— This is a nitrog- 
enous compound of simpler constitution than proteids. It 
is formed within plants both analytically by the decomposi- 
tion of proteid, and synthetically probably by the combination 
of simpler substances. Asparagin is soluble in water and in 
the cell-sap, and is one of the most important nitrogenous 
compounds capable of solution and circulation within plants. 
It combines with non-nitrogenous compounds to form pro- 



ASPARAGIN — BASSORIN 



303 



teids, and is apt to accumulate in those parts of plants where 
there is not sufficient non-nitrogenous material at hand for 

the formation of proteids. The accumulation of asparagin is 
particularly apt to occur in plants which arc grown in the 
dark, so that carbon assimilation does not take place. Thus, 
Pfeffer found that when seedlings of lupin were grown in the 
dark, they contained a large amount of asparagin, but when 
they were brought to the light, the asparagin disappeared. 
He found that this was not due simply to the influence of the 
light, for when the seedlings were exposed to the light in an 
atmosphere destitute of carbon dioxide, the asparagin per- 
sisted in the seedlings. For the ready demonstration of as- 
paragin. tubers of Dahlia may be employed. Rather thick 
sections are cut from a tuber while the razor is kept dry and 
transferred to a few drops of alcohol on a glass slide and 
covered with a coverglass. On the evaporation of the alco- 
hol crystals of asparagin in the form of rhombic plates are 
deposited on the coverglass and slide. To determine whether 
the crystals are asparagin, they are treated with a few drops 
of an entirely saturated solution of asparagin, which must 
be of the same temperature as the preparation. If the 
crystals are asparagin, instead of being dissolved they will 
increase in size, while other substances than asparagin will 
dissolve in the saturated asparagin solution just as they would 
in water. It is characteristic of asparagin that if the crystals 
are heated to ioo° C, they lose their water of crystallization 
and appear like bright droplets of oil. At 200 asparagin 
becomes decomposed and forms frothy brown droplets which 
are no longer soluble in water. 

Atropine, C 17 H 23 N0 3 . — This alkaloid with its isomers 
hyoscyamin, pseudohyoscyamin, and hyoscin, occurs widely 
distributed in the Solanacese. Sections of roots of Atropa 
Belladonna contain atropine and yield a brownish precipitate 
when treated with potassium iodide-iodine. 

Bassorin. — Gum tragacanth, obtained from certain cells of 
the pith and medullary rays of several species of Astragalus. 



304 MICROCHEMISTRY OF PLANT PRODUCTS 

Swells strongly in water, but does not go into complete solu- 
tion. Is not colored either by iodine or chloroiodide of zinc. 

Berberin, C 20 H 17 NO 4 + 6H 2 0. — This yellow alkaloid oc- 
curs in the young parenchymatous tissue, and in the older 
xylem portions of Berberis vulgaris, and in representatives of 
the most various families. With potassium iodide-iodine it 
forms a reddish-brown precipitate which, by treatment with 
alcoholic potassium iodide-iodine, becomes changed into tubu- 
lar or hair-like forms having a brownish or iridescent green 
color. Ammonium and nitric acid impart to berberin a red- 
dish-brown color. A solution of potassium bichromate or 
potassium iodide in 50 per cent, sulphuric acid produces, with 
berberin, an intense purplish-red color. One part of nitric 
acid mixed with 100 parts of water added to sections contain- 
ing berberin will produce clustered acicular crystals of ber- 
berin nitrate within the berberin-bearing cells. 

Betulin, C 14 H 18 8 + H 2 0. — This glucoside occurs in the 
form of fine granules in the thinner walled cork cells of birch 
bark. It is accompanied by the enzyme betulase, which splits 
it into glucose and methylsalicinic ester. In order that it may 
be studied to good advantage under the microscope, the air 
should be pumped from sections immersed in water, and then 
the sections should be examined in water under the microscope. 
Betulin is insoluble in water, but is soluble in alcohol. It is 
strongly antiseptic, and protects birch bark against the attacks 
of lower organisms. 

Betuloretic Acid, C 36 H 66 5 . — This is secreted by the 
glandular hairs on the leaves of Betula alba. It is obtained 
from the thick, pale yellow secretion by successive solution in 
boiling alcohol, ether, and an aqueous solution of sodium car- 
bonate. It is colored a beautiful red by concentrated sulphuric 
acid. 

Brucine, C 23 H 26 N 2 4 + 4H 2 0. — The alkaloid brucine oc- 
curs along with strychnine in the seeds of various species of 
Strychnos. Ammonium vanadnate in sulphuric acid gives 
with brucine a yellowish-red color. When sections containing 



CAFFEINE — CALCIUM CARBONATE 305 

brucine are treated with a mixture of nitric and hydrochloric 
acids, the cell-contents are colored a reddish-orange, which 
merges into yellow. 

Caffeine, C s H 10 N 4 O 2 + H 2 0. — The narcotic alkaloid in 
many foods and drugs. It occurs in plants of various fami- 
lies; for instance, in Thea, Coffea, Theobroma, Cola, Ilex, 
Sterculea, Neea. When sections containing caffeine (theine, 
methyl-theobromine, trimethyl-xanthin) are treated with a 
drop of concentrated hydrochloric acid, and then after a 
minute with a drop of a 3 per cent, gold chloride solution, 
somewhat slender, yellowish, silken crystals of a double chlo- 
ride of gold and caffeine begin to be formed on the evapora- 
tion of the reagent. However, theobromine forms quite simi- 
lar crystals when treated as above. Another method for the 
detection of caffeine is to place sections in a few drops of 
water and heat to boiling; then to allow the water to evapo- 
rate slowly and to treat the residue with a drop of benzol. On 
the evaporation of the benzol, caffeine appears in the form of 
fine needle-crystals. 

Calcium. — When the ash of plants is treated with sulphuric 
acid, this unites with the calcium present to form crystals of 
gypsum. If calcium sulphate is already present in the ash, its 
characteristic crystals may be detected when an aqueous solu- 
tion of the ash is allowed to dry slowly. If calcium is present 
in sections, it may be deposited in the form of crystals of cal- 
cium oxalate if the sections are treated with a solution of 
ammonium oxalate. 

Calcium Carbonate, CaC0 3 . — This rarely occurs in the 
crystalline form within the cells. It may, however, be found 
imbedded in, or incrusted on, the cell-walls. Calcium carbon- 
ate dissolves with effervescence when treated with dilute acetic 
acid. When treated with concentrated hydrochloric acid, it 
dissolves with the evolution of carbon dioxide gas. The in- 
growths from the walls of certain cells of the leaves of Ficus 
elastica, known as cystoliths, are thickly incrusted with cal- 



306 MICROCHEMISTRY OF PLANT PRODUCTS 

cium carbonate and afford excellent material for the demon- 
stration of this salt within plant tissues. 

Calcium Phosphate, Ca 3 (P0 4 ) 2 . — This salt of calcium oc- 
curs usually, if not always, in solution in the cell-sap. It may 
be deposited in the form of sphaerocrystals when plant tissues 
containing it are kept for a long time in strong alcohol. When 
treated with sulphuric acid, the sphaerocrystals are dissolved 
and crystals of calcium sulphate are formed in their stead. 
When sections containing calcium phosphate are heated on a 
slide in a drop of ammonium molybdate acidulated with nitric 
acid, a yellow precipitate is produced. This reaction may be 
hindered by the presence of certain organic compounds, such 
as potassium tartrate, in which case the sections should be 
treated with a mixture of 25 volumes of a concentrated aque- 
ous solution of magnesium sulphate with 2 volumes of a con- 
centrated aqueous solution of ammonium chloride and 15 vol- 
umes of water. In this case a crystalline precipitate of am- 
monio-magnesium phosphate is formed. 

Calcium Oxalate, CaC 2 4 . — Crystals of calcium oxalate 
occur so commonly in plants that it is safe to assume that any 
crystals observed in fresh tissues are of this substance until 
the contrary is demonstrated. The crystals may occur singly 
in the cells, in which case their definite crystalline form can 
be made out, or in the form of agglomerated star-shaped clus- 
ters of crystals, or in bundles of parallel needle-shaped crys- 
tals, or they may occur very numerously in cells in the form 
of very minute crystals. The crystals are insoluble in water 
and acetic acid, but dissolve without effervescence in hydro- 
chloric acid. When they are treated with sulphuric acid, crys- 
tals of calcium sulphate are formed in their place. Calcium 
oxalate appears to be an excretion formed by the union of 
salts of calcium, which have been absorbed from the soil, with 
oxalic acid which is formed by the plant. 

Calcium Sulphate, CaS0 4 . — Minute crystals of calcium 
sulphate occur in many desmids. They are insoluble in con- 



CALLOSE — CANB-SUGAB 3°7 

centrated sulphuric acid. A solution of barium chloride dis- 
solves them with the formation of barium sulphate. 

Callose. — The chemical nature of callose is not precisely 
known ; it is supposed by some to be a proteid. Callose occurs 
in sieve tubes, where it may close up the sieve pores. It also 
occurs commonly in cystoliths, and in the membranes of pol- 
len-brains and various fungi. Callose is insoluble in water, 
alcohol, and cuprammonia, but it is readily soluble in cold 
sulphuric acid, calcium chloride, and concentrated chloride of 
zinc. It is insoluble in cold alkaline carbonates, but swells up 
without dissolving in ammonium. Corallin, aniline blue, and 
a mixture of soluble blue and vesuvin, or of vesuvin and orseil- 
lin, are suitable stains for callose. The corallin should be 
dissolved in a saturated solution of sodium carbonate. After 
remaining in this solution for a time, the sections should be 
examined in glycerine. If the sections are overstained, the 
intensity of the stain may be reduced in a 4 per cent, solution 
'of sodium carbonate. The aniline blue should be used in 
dilute aqueous solutions, in which the sections are to remain 
for about half an hour. Overstaining may be remedied by 
washing out in glycerine. 

Calycin, C 18 H 12 5 . — This occurs in the tissues of many 
lichens. Its presence may be demonstrated by moistening 
some of the powdered lichen with glacial acetic acid, and when 
the preparation dries, the long, doubly refractive crystals of 
calycin are deposited. When a section of lichen containing 
calycin is treated on the slide with a few drops of chloroform 
and a drop of sodium hydrate, that portion of the section 
which contains calycin assumes a color varying from brick ^ 
red to blue-red. 

Cane-sugar (Sucrose), C 12 H 22 O l:l . — This carbohydrate is 
of common occurrence in plant tissues. At 15 C. it is solu- 
ble in J part of water. It is difficultly soluble in alcohol. 
When boiled with Fehling's solution, it does not at first pre- 
cipitate cuprous oxide, but on longer boiling it becomes con- 
verted into glucose and laevulose, which are capable of reduc- 



308 MICROCHEMISTRY OF PLANT PRODUCTS 

ing Fehling's solution. If rather thick sections containing 
cane-sugar (the sugar-beet affords good material) are placed 
for a short time in a concentrated solution of cupric sulphate, 
and then quickly rinsed in water, transferred to a solution of 
I part of potassium hydrate in i part of water, and heated to 
boiling, the cells containing the sugar take on a sky-blue color. 
A blue color is also produced by Fehling's solution when sec- 
tions containing cane-sugar are heated in a drop of the solu- 
tion on a slide until bubbles arise. 

Carotin, C 2G H 38 . — Carotin occurs in the orange and red 
chromatophores of many flowers and fruits, and, indeed, most 
orange and red colors of both plants and animals seem to 
belong to the carotins; carotin seems also to be an essential 
part of chlorophyll; it occurs in crystalline form in the roots 
of carrots, which have a yellow color in consequence. To 
demonstrate the presence of carotin in chloroplasts place pieces 
of fresh leaves in a 20 per cent, solution of potassium hydrox- 
ide in 40 per cent, alcohol, and leave them thus in a tightly' 
closed vessel for several days. When the chlorophyll has 
been extracted from the leaves, they should be washed in dis- 
tilled water and sections from them should be mounted in 
glycerine. Yellowish and red crystals will then be found in 
the cells which formerly contained chlorophyll. Carotin is 
insoluble in water and with difficulty in alcohol, but is readily 
soluble in petroleum ether, benzol and benzine. When freshly 
dried leaves or roots of carrots are powdered and treated with 
one of these solvents, and the solution is allowed to dry or it is 
treated with alcohol, carotin crystallizes out in the form of 
reddish or yellowish crystals. With a solution of iodine car- 
otin is colored greenish or bluish ; with concentrated sulphuric 
acid it is colored from violet to indigo blue. 

Cellulose, C 6 H 10 O 5 . — Cellulose is one of the most impor- 
tant constituents of cell- walls ; the first- formed walls are nearly 
always of cellulose, together with certain pectic compounds. 
Modified cell-walls — namely, those which have become cutin- 
ized or lignified — have arisen by the chemical modification of 



CHITIN COLCHICINE 309 

cellulose, or by the infiltration of new material between the 
cellulose molecules, or by both of these processes. Cellulose 
is characterized by being soluble in sulphuric acid and cupram- 
monia ; by being colored from violet to blue by sulphuric acid 
and iodine, chloroiodide of zinc, chloroiodide of calcium, iodine 
and aluminum chloride, iodine and phosphoric acid. See under 
these heads in the chapter on Reagents. 

Chitin. — The chemical composition of chitin is not pre- 
cisely known, but it has been estimated to be C 18 H 30 N 2 O 12 . 
The walls of many fungi consist of chitin instead of cellu- 
lose. This may be demonstrated by cutting the pileus of an 
Agaricus into small pieces, which are then to be treated suc- 
cessively with dilute potassium hydrate, dilute sulphuric acid 
heated to boiling, alcohol, and finally ether. When this 
process is completed, a white substance remains which becomes 
hard and horny on drying, and which is insoluble to all rea- 
gents except concentrated acids, and in all other respects pos- 
sesses the characteristics of chitin. 

Chlorophyll, possibly C 10 H 20 NPO 9 . — Chlorophyll may be 
extracted from the chloroplasts by means of strong alcohol. 
When this extract is shaken up with benzole and a few drops 
of water, and allowed to stand for a short time, the benzol 
which rises to the top will contain two pigments, amorphous 
chlorophyll-green and carotin; while the lower stratum of 
alcohol will contain a crystallizable chlorophyll-green and 
xanthophyll. The amorphous and the crystallizable chloro- 
phyll-green differ in the character of their spectra and in their 
solubility in different reagents. The amorphous form is solu- 
ble in alcohol, petroleum ether, carbon bisulphide and benzine ; 
while the crystallizable is soluble only in the alcohol. 

Coffee-tannin, C 15 H 18 8 . — This occurs in the endosperm 
of the coffee-bean. Its presence is indicated when sections 
give an abundant precipitate with lead acetate, a deep yellow 
color with ammonia and caustic potash, and a dark green 
color with ferric chloride. 

Colchicine, C 22 H 25 NO G . — This occurs in a few rows of 



3IO MICROCHEMISTRY OF PLANT PRODUCTS 

cells immediately surrounding the vascular bundles of the 
corm of Colchicum autumnale. Treated with a mixture of i 
part of sulphuric acid and 3 parts of water colchicine is col- 
ored yellow, and this color is changed to a brownish-violet by 
the addition of a crystal of potassium nitrate. Iodine stains 
it brown, and potassic-mercuric iodide and hydrochloric acid 
produce with it a yellow precipitate. 

Corydalin, C 22 H 27 N0 4 . — This is an alkaloid which is found 
in the idioblasts of the Fumariacese. When corydalin is pres- 
ent, ammonia produces a dark gray precipitate, picric acid a 
yellow, and potassium iodide-iodine a deep reddish-brown 
precipitate. 

Crocin (Saffron-yellow), C 44 H 70 O 28 . — This is a glucoside 
occurring in the stigmas of Crocus sativus. When concen- 
trated sulphuric acid is added to crocin, a deep blue color is 
produced which passes into violet, cherry-red and then brown. 
Nitric acid also produces a blue color which passes into brown. 

Curarin. — This alkaloid occurs in the parenchyma and bast 
of several species of Strychnos. Concentrated nitric acid pro- 
duces with it a blood-red, and dilute or concentrated sulphuric 
acid a carmine-red color. 

Curcumin, C 14 H 14 4 . — Curcumin occurs, dissolved in an 
ethereal oil, in certain cells of the ground parenchyma of the 
rhizome of species of Curcuma and probably in other members 
of the Zingiberaceae family. It crystallizes in the form of 
yellow needles which have a bluish tint by 'reflected light. 
Lead acetate forms a brick-red precipitate with curcumin, and 
sulphuric acid gives it a crimson color. 

Cutin. — Cutin is a substance which is nearly related to 
suberin (which see), but is not identical with it. None of the 
acids derived from cutin is identical with any derived from 
suberin. However, the micro-chemical reactions of suberin 
and cutin are the same. They are insoluble in concentrated 
sulphuric acid and cuprammonia, and are colored from yellow 
to brown with the iodine reagents. When heated with con- 
centrated potassium hydrate, they form yellowish droplets and 



CYTISIN — DIASTASE 3 I I 

granular masses. When heated in nitric acid and potassium 
hydrate, they form droplets which melt between 30 and 40 
C, and which are soluble in boiling alcohol, ether, benzol, 
cliloroform and dilute potassium hydrate. Both suberized and 
cutinized walls resist concentrated chromic acid at ordinary 
temperatures. Chemical analysis shows that cutin is com- 
posed of compound esters and fatty acids, and when heated to 
300 in glycerine, it behaves as a fatty body. For staining 
cutinized walls, see under Cyanin, Alcannin, Chlorophyll 
Solution. 

Cytisin, C n H 14 N 2 0. — This alkaloid occurs in the seeds of 
Cytisus laburnum and of other species of Cytisus and in spe- 
cies of Laburnum, Genista, Ulex, Sophora, Thermopsis, Bap- 
tisia, Anagyris, Lotus, Colutea and Euchresta. It occurs in 
less abundance in other parts of the plant, such as the petals 
and peripheral tissues of the stem. Potassium iodide-iodine 
produces with it a reddish-brown, granular precipitate which 
is soluble in sodium hyposulphite. Chloride of iron gives an 
orange-red color with cytisin. With phospho-molybdic acid 
a light yellow precipitate is produced, and picric acid when 
added to thin sections containing cytisin produces crystal 
groups of a reddish-yellow color. 

Datiscin, C 2 iH 22 12 . — This glucoside is found in the cell- 
walls of the wood and bark of Datisca cannabina. Lime and 
baryta waters produce with it a yellow solution which loses its 
color on the addition of acetic or dilute hydrochloric acid. In 
the presence of datiscin, acetate of lead and chloride of zinc 
produce a yellow, oxides of copper a greenish, and chloride of 
iron a dark bluish-green, precipitate. 

Dextrine, C 12 H 20 O 10 . — This carbohydrate is one of the 
intermediate products between starch and maltose (see Amy- 
lose). It is easily soluble in water, and from its aqueous solu- 
tion it may be precipitated by strong alcohol. It is not col- 
ored blue by iodine, and does not reduce salts of copper. 

Dextrose (Glucose, Grape-sugar). — See under Glucose. 

Diastase. — To demonstrate the presence of diastase in sec- 



312 MICROCHEMISTRY OF PLANT PRODUCTS 

tions they are laid for a time in a dark brown solution of 
guaiacum in absolute alcohol. When the sections are com- 
pletely infiltrated with this solution the alcohol is allowed to 
evaporate, and then the sections are placed in a rather dilute 
solution of hydrogen peroxide. By this treatment cells con- 
taining diastase are colored a beautiful blue. See also under 
Diastase Solution in the last chapter. 

Dulcite, C 6 H 14 6 . — Dulcite may be demonstrated in sec- 
tions from one-year-old stems of Euonymus japonicus. The 
sections are placed on a slide in a few drops of alcohol, and 
covered with a coverglass. After the alcohol has slowly 
evaporated from under the coverglass, crystals of dulcite will 
be deposited in the form of long, branched prisms or needles 
radiating from a common center. They are distinguished 
from crystals of potassium nitrate by dissolving in diphenyla- 
mine and sulphuric acid without coloration, and by being 
insoluble in a concentrated solution of dulcite. 

Elaioplasts. — These are rounded or irregularly polygonal, 
more or less granular bodies, consisting of a protoplasmic 
stroma and inclosed oil, which occur closely applied to the 
nucleus in the epidermal cells of many monocotyledonous and 
some dicotyledonous plants. In old cells the elaioplasts have 
the appearance of a sponge saturated with oil. The oil in the 
elaioplast of Ornithogalum umbellatum may be almost in- 
stantly dissolved by means of alcohol. The elaioplasts may 
be fixed and stained at the same time by treating sections con- 
taining them with a dilute solution of alcannin in i per cent, 
acetic or formic acid. The acid fixes the protoplasmic stroma, 
while the alcannin stains the oil a beautiful red. The fixing 
and staining process should be complete in five minutes. If 
desired, the sections may be double-stained by transferring 
them from the alcannin to a solution of iodine-green and 
glycerine, after which they may be mounted in glycerine- jelly. 
The sections may also be stained in a mixture of a dilute solu- 
tion of alcannin and a solution of iodine-green in 50 per cent, 
alcohol and 1 per cent, acetic acid. 



EMULSIN — FATS 3 I 3 

Emulsin. — This is a glucoside-splitting ferment which oc- 
curs in certain cells of the almond and of the bundle-sheath 
of the leaves of Prunus laurocerasus and in other Rosacese, 
whore it splits up the glucoside amygdalin into glucose, hydro- 
cyanic acid and benzaldehyde. When sections are treated with 
Millon's reagent, the cells containing emulsin take on an 
orange-red color, while the surrounding cells are colored a 
pale rose-red. A solution of copper sulphate and caustic 
potash produces a violet color in the emulsin-bearing cells. 

Ethereal Oils. — Ethereal oils are distinguished from fatty 
oils in that they may be distilled from plants along with vapor 
of water, and are soluble in glacial acetic acid, and an aqueous 
solution of chloral hydrate. At 130 C. all ethereal oils may 
be driven from sections, while the fatty oils remain behind. 
Ethereal oils are only slightly soluble in water, but they impart 
their smell strongly to it. They are easily soluble in ether, 
chloroform and fatty oils. The spot produced on paper by 
ethereal oils soon disappears. They agree with the fatty oils 
in being browned or blackened by osmic acid, and in being 
stained by alcannin and cyanin. 

Eugenol, C G H 3 .OH.OCH 3 .C 3 H 5 . — Eugenol occurs in clove 
and pimento oil. When sections containing either of these 
oils are treated with a concentrated solution of potassium 
hydrate, long columnar or needle-shaped crystals of potassium 
caryophyllate are produced. When sections of cloves are 
used, they often become covered by the forming crystals. 

Fats and Fatty Oils. — These are insoluble in cold and hot 
water, and, with the exception of castor oil, hardly soluble in 
alcohol, but readily soluble in ether, chloroform, benzol, 
ethereal oils, aceton and wood spirit. They make a spot on 
paper which does not disappear, as in the case of ethereal oils. 
Most fats and fatty oils are colored brown or black by 1 per 
cent, osmic acid. When a drop of fat or fatty oil is placed 
on a glass slide in a drop of a mixture of equal parts of con- 
centrated potassium hydrate and ammonium, the oil becomes 
saponified, and may assume a form like a bunch of grapes, 



314 m M1CR0CHEMISTRY OF PLANT PRODUCTS 

or it may be partly or wholly changed into clusters of soap 
crystals. Vapor of hydrochloric acid has been used to dis- 
tinguish between ethereal and fatty oils. A large and a small 
glass ring, such as are used for hanging-drop cultures, are 
cemented to a glass slide, the small one being shallower than 
the large one, and placed within it concentrically. Hydro- 
chloric acid is placed into the space between the rings, and the 
sections to be tested are placed on a coverglass in a drop of 
glycerine containing a large amount of sugar. 

The coverglass is then inverted and placed on the larger 
ring. After the vapor of hydrochloric acid has had time to 
act, any ethereal oil present in the sections will take on the 
form of bright yellow drops which finally disappear. Fatty 
oils do not form yellow drops by this treatment. A solution 
of alcannin colors the fats red, but several hours may be 
required to accomplish this. A solution of cyanin in 50 per 
cent, alcohol is also a good stain for fats. The sections will 
not need to lie in this stain longer than half an hour. If the 
sections are overstained they may be washed out in glycerine 
or a concentrated solution of potassium hydrate. 

Frangulin, C 20 H 20 O 10 . — This glucoside occurs in the cortex 
of species of Rhamnus. It forms yellow crystalline masses 
which are insoluble in water, but soluble in alkalies, which pro- 
duce with it a cherry-red color. Concentrated sulphuric acid 
produces with frangulin an emerald-green, which changes into 
purple, and finally the frangulin dissolves with a dark red 
color. Water will precipitate it from this solution. 

Fungus Cellulose. — The membranes of very few fungi 
give the reactions of cellulose. The membranes of most fungi 
are insoluble in cuprammonia, and are colored from yellow to 
brown by chloroiodide of zinc, sulphuric acid and iodine. 
Neither do they react in the same manner as suberized and 
lignified membranes. They are, therefore, considered to be 
a distinct substance, which is termed fungus cellulose. See 
also under Chitin. 

Gelatinous Sheaths. — The homogeneous gelatinous sheaths 



GELATINOUS SHEATHS GLOBOIDS 315 

which cover the entire outside of certain algae — notably, species 
of Spirogyra and Zygonema — may be demonstrated by the use 
of certain stains and other substances, such as India ink, which 
may become deposited in the sheaths. Aqueous solutions of 
vesuvin, methyl-violet and methylene-blne will stain both the 
cell-walls and gelatinous sheaths, but the latter with less inten- 
sity. Chloroiodide of zinc will stain the wall without affecting 
the sheaths. Turnbull's blue may be deposited in the gela- 
tinous sheaths in the following manner: A small number of 
Zygnema filaments, for instance, may be tied together with a 
thread and placed for about two minutes in a 2 per cent, solu- 
tion of ferrous lactate, then quickly washed in water, and 
transferred to a 0.2 per cent, solution of ferricyanide of potas- 
sium. A small amount of Turnbull's blue will then be depos- 
ited in the gelatinous sheaths. This process should be repeated 
several times, until the deposit of Turnbull's blue is sufficiently 
dense to cause the sheaths to stand out quite sharply. By this 
method very instructive double stains may be achieved with 
algae which have been growing in a dilute solution of Congo- 
red (see under this head in the last chapter), which stains the 
cell-walls, but not the gelatinous sheaths. See also in the last 
chapter under India Ink. 

Globoids. — The globoids found in aleurone grains consist 
of a double phosphate of calcium and magnesium, which is 
insoluble in alcohol and dilute potassium hydrate, but is soluble 
in dilute mineral acids and in acetic, oxalic and tartaric acids. 
In an ammoniacal solution of ammonium phosphate the glo- 
boids are replaced by groups of crystals of ammonium-mag- 
nesium phosphate. Treated with ammonium oxalate, they 
become replaced by crystals of calcium oxalate. The globoids 
may be isolated to a certain extent by extracting the oil from 
sections of endosperm containing them by means of alcohol 
or alcohol and ether, and then dissolving the ground substance 
and crystalloid by means of a 1 per cent, potassium hydrate. 
If crystals of calcium oxalate are present along with the 



3l6 MICROCHEMISTRY OF PLANT PRODUCTS 

globoids, they may be distinguished by means of the polarizer, 
since they are doubly refractive, while the globoids are not. 

Glucose, C 6 H 12 6 . — This carbohydrate occurs in sweet 
fruits and in the leaves and other members of plants, being 
one of the most common forms in which carbohydrates circu- 
late within the plant. The warty crystals of glucose which 
are deposited from aqueous and alcoholic solutions at low tem- 
peratures melt at 86°, and become free from water at no° 
C. At from 30 to 35 ° C. glucose crystallizes from concen- 
trated solutions in water, ethyl- and methyl-alcohol in the 
form of hard crusts, which melt at 146 C. The presence of 
glucose may be easily demonstrated in the fruit of the pear, 
for instance, and in the leaves of Balsamina, or other rather 
translucent leaves which have been cut from the parent plant 
and kept fresh under a bell-jar for several days. Pieces of 
the flesh of a ripe pear may be put into a test-tube with 
Fehling's solution and brought to a boil, when a reddish pre- 
cipitate of cuprous oxide will be thrown down. This reaction 
is characteristic of dextrose, maltose, lactose, lsevulose and 
many glucosides. In this instance, however, we are dealing 
with dextrose. This reaction may also be carried out on the 
microscope slide. Sections from the pear three or four cell- 
layers thick should be placed on the slide in a few drops of the 
solution, the coverglass should then be put on, and the solu- 
tion heated until bubbles begin to arise. The microscope will 
then reveal the granular precipitate of cuprous oxide within 
the cells. Portions of the leaf of the Balsamina may be 
treated on the slide as directed for the sections from the pear. 
See under Fehling's Solution in the last chapter. 

Glycogen, C 6 H 10 O 5 . — This is a colorless, amorphous, 
highly refractive substance occurring quite commonly in the 
cells of fungi. It is soluble in water, but within the cells it 
may be stained a reddish-brown by means of iodine. 

Gums. — These are amorphous, transparent substances 
which dissolve in water more or less completely and form a 
sticky solution. They may be precipitated from their aqueous 



GUMS HESPERIDIN 3 I 7 

solutions by alcohol. Those glims which dissolve in water 
completely, such as the gum of the cherry, apricot, peach and 
gum arabic, are called true gums, while those which contain 
cellulose and are not completely soluble in water are known as 
mixed gums. Gum tragacanth is an example. One of the 
most striking characteristics of gums which may be used in 
their identification is their great capacity to swell in water. 
To follow the process of swelling with the microscope, sec- 
tions should be cut from dry material with a razor which may 
be wetted with alcohol, but not with water. The sections 
should be placed on a slide in a drop of strong alcohol, the 
coverglass should be put on, and a drop of water placed on 
the slide so that it just touches the edge of the coverglass. 
As the water mixes with the alcohol and comes in contact with 
the section a slow swelling of 'the gum will begin, which may 
be followed very accurately through the microscope. For 
directions for staining see under Mucilage, and for making 
permanent preparations of sections containing mucilages and 
gums see under Boracic Acid in the last chapter. 

Hemicelluloses. — These are carbohydrate reserve materials 
which are deposited as additions to the cell-walls in the endo- 
sperm of seeds and in w T ood parenchyma and wood-fibers. By 
means of enzymes they may be converted into gums and 
sugars, in which forms they may be transported to those parts 
where growth is taking place. The hemicellulose or reserve 
cellulose in the endosperm of the date seed acts like ordinary 
cellulose in being colored blue by chloroiodide of zinc and in 
dissolving in cuprammonia. The reserve cellulose in the en- 
dosperm of the seeds of Lupinus luteus is not dissolved in 
cuprammonia, and does not assume a blue color when treated 
with chloroiodide of zinc. 

Hesperidin, C 21 H 2G 12 . — This glucoside occurs dissolved 
in the cell-sap of many plants. It may be precipitated from its 
solution in the cell-sap by means of alcohol. The precipitate 
is in the form of crystals, which are colorless or slightly yel- 
low, and are doubly refractive, so that they may be studied to 



3l8 MICROCHEMISTRY OF PLANT PRODUCTS 

good advantage by means of the polarizer. Hesperidin is 
also precipitated on the drying up of the cell-sap. The crys- 
tals of hesperidin are insoluble in cold and boiling water, alco- 
hol, ether, benzine and dilute acids, but they are soluble in 
solutions of caustic potash and soda, and in ammonia, yielding 
a yellowish color to the solvent. Hesperidin may readily be 
obtained for study in the unripe fruit of the orange and in 
the epidermal cells of Capsella bursa-pastoris. Hesperidin 
may become deposited in the form of sphaerocrystals, when 
the tissues containing it have lain for some time in strong 
alcohol or glycerine, acting in this respect similarly to .inulin. 
The constituent acicular crystals of the hesperidin sphserites 
can be more easily distinguished than those of inulin, and 
when the hesperidin sphserites are treated with a drop of 
a-naphtol, and then with two or three drops of concentrated 
sulphuric acid, they dissolve with a yellow color, while, with 
like treatment, inulin sphaerites dissolve with a violet color. 

Indican. — The glucoside indican is a substance of the con- 
sistency of syrup, and of a yellowish or brownish color. It 
is found in Isatis tinctoria, Phajus grandifolius, and in other 
indigo-bearing plants. When tissues containing indican are 
exposed to the air, they may take on a blue color due to the 
conversion of the indican to indigotin, which may be precipi- 
tated in alcohol in the form of small, tabular, bluish crystals. 
To demonstrate the presence of indican, tissues containing it 
should be placed under a bell-jar and over a dish of absolute 
alcohol. After standing exposed to the vapor of alcohol for 
twenty- four hours, the tissues will be colored blue by the indigo 
blue which will have been formed from the indican. A piece 
of moistened filter-paper should be placed under the bell-jar 
to keep the tissues from drying. 

Inulin, C 12 H 20 O 10 . — Inulin is a carbohydrate which occurs 
dissolved in the cell-sap of many plants, particularly among 
the Composite. It may be deposited from its solution in the 
cell-sap by means of alcohol. To study the sphaerocrystals of 
inulin, pieces of dandelion or Dahlia roots should be placed in 



INULIN LIGNIFIED MEMBRANES 3 I 9 

50 per cent, alcohol for a week or more, and then thin sections 
should be prepared and examined in a drop of the alcohol 
under the microscope. The sections should nol be placed in 
water, since the crystals of inulin are soluble in it. The 
sphaerites will appear applied to the walls of the cells as shown 
in Fig. 98. When the alcohol is replaced by water which is 
then heated over a flame, the sphaerites will dissolve. If sec- 
tions containing inulin sphaerites are treated with a 20 per 
cent, solution of a-naphtol, and then 2 or 3 drops of concen- 
trated sulphuric acid are added, the sphaerites will be seen to 
dissolve with a violet color. Inulin does not reduce Fehling's 
solution. 

Leucin, C 5 H 10 NH 2 .COOH. — Leucin belongs to the amido- 
compounds. It has been found in the etiolated leaves of Pas- 
palum elegans and Dahlia variabilis, and associated with as- 
paragin in seedlings of various Leguminosae, particularly in 
those of Lupinus. Leucin crystallizes in thin plates, which are 
lighter than water and have the appearance of mother-of-pearl. 
If sections containing leucin are carefully heated on a slide 
under a coverglass to a temperature of 170 C, the cover- 
glass will become covered with minute, scale-like crystals, 
which are doubly refractive and may be studied to advantage 
by means of the polarizer. The crystals of leucin may also 
be obtained if sections are treated with alcohol under a cover- 
glass, and the alcohol is then allowed to slowly evaporate. 

Leucoplasts. — For methods of fixing and staining leuco- 
plasts, see in the last chapter under Acid Fuchsin, Gold Chlo- 
ride, and Picronigrosin. 

Lignified Membranes. — Lignified membranes are distin- 
guished from cellulose membranes by their insolubility in 
cuprammonia, and by being colored from yellow to brown by 
iodine or chloroiodide of zinc. One of the most reliable tests 
for lignified membranes will be found in the last chapter under 
Phloroglucin. Aniline sulphate is also a good test for lig- 
nified membranes. The sections are first mounted in a drop 
of a concentrated solution of aniline sulphate, and then this 



320 MICROCHEMISTRY OF PLANT PRODUCTS 

'is replaced by a drop of concentrated sulphuric acid. By this 
treatment lignified membranes are stained a golden yellow. 

Lipochromes. — These are yellow and red pigments which 
are for the most part dissolved in fatty substances within the 
cells, and which are colored blue by sulphuric or nitric acid, 
and green by potassium iodide-iodine. 

Magnesium. — To demonstrate the presence of magnesium 
within plant tissues, sections are placed on the slide in a drop 
of a solution of sodium phosphate or sodium-ammonium phos- 
phate, and a little ammonium is added. In the presence of 
magnesium, crystals of ammonio-magnesium phosphate are 
then formed, which have a coffin-lid form. When the ash 
of tissues containing magnesium is treated as above, the crys- 
tals are apt to form in x- or ^-shaped groups. 

Maltose. — Maltose is a sugar which is produced from starch 
by the action of diastase. Maltose reduces Fehling's solution, 
but only about two thirds as much as does grape-sugar (dex- 
trose, glucose). 

Morphine, C 17 H 19 N0 3 -f H 2 0. — This was the first alka- 
loid to be extracted pure and studied. It is the chief alkaloid 
in opium obtained from the latex of Papaver somniferum. 
When the latex containing morphine is treated with potassium 
iodide-iodine, a reddish-brown precipitate is produced, with po- 
tassium-bismuth iodide a reddish-orange, and with potassium- 
mercuric iodide a yellowish-white, precipitate, while phospho- 
molybdic acid produces a yellow precipitate. A solution of 
5 drops of methylal in I c.c. of concentrated sulphuric acid 
gives a violet color to latex containing morphine. Codeine, 
C 18 H 21 N0 3 , associated with morphine in opium, has essen- 
tially the same color reactions. On treating opium infusion 
with ammonia, morphine is precipitated and codeine is left in 
solution. 

Mucilages (see also under Gums). — Mucilage contained in 
sections of plant tissues may be differentiated by staining with 
methylene-blue. Place the sections in a deep blue solution of 
methylene-blue in equal parts of alcohol, glycerine and water. 



MUCILAGES NICOTINE 32 1 

This operation may be done on the glass slide. The staining 
will soon be completed and the sections will then be ready for 
examination under a coverglass. If the sections are taken 
from fresh materials, the razor should be moistened with alco- 
hol. Dry materials should be soaked in water to soften for 
cutting. If it is found that the mucilage dissolves too much 
in the water, the mucilage may be hardened and the tissues 
softened at the same time in the lead acetate solution described 
under Gums. 

Mustard Oil, C 3 H 5 CNS. — Seeds and the vegetative organs 
of the Cruciferae, Resedaceae, Capparidaceae, Tropaeolaceae, 
and Lemnanthaceae contain peculiar nitrogenous glucosides 
which become decomposed into sulphur-bearing substances, 
longjoiown as mustard oils, by means of the enzyme myrosin. 

Myrosin. — Myrosin is an enzyme occurring in certain spe- 
cialized cells in the seeds and other parts of many Cruciferae, 
etc. The cells containing myrosin are stained a deep red by 
Millon's reagent, while the surrounding cells may be stained 
a pale rose color. When sections containing myrosin are 
heated in a concentrated solution of hydrochloric acid which 
contains a drop of a 10 per cent, aqueous solution of orcin in 
each cubic centimeter, a violet color is produced in the cells 
containing the myrosin. Myrosin produces allylic mustard 
oil from potassium myronate, a glucoside which occurs in the 
parenchyma cells which are associated with those containing 
myrosin. 

Narceine, C 23 H 27 NO s + 3H 2 0. — This is an alkaloid oc- 
curring associated with morphine in the latex of Papaver som- 
niferum. When a yellow color follows the addition of meth- 
ylal to the latex, the presence of narceine is indicated. 

Narcotine, C 2 2H 23 N0 7 . — An alkaloid associated with mor- 
phine in opium. Sodium selenate produces with it an orange- 
red color. 

Nicotine, C 10 H 14 N 2 . — This alkaloid occurs in most species 
of Nicotiana, especially in N. Tabacum. It has not been found 

22 



322 MICROCHEMISTRY OF PLANT PRODUCTS 

outside this genus. When sections containing nicotine are 
treated with potassio-mercuric chloride, a yellowish-white pre- 
cipitate is produced. Phospho-molybdic acid gives, with nico- 
tine, an abundant yellow precipitate. In the presence of nico- 
tine mercuric chloride produces a white, and platinum chlo- 
ride a yellow, precipitate, while potassium iodide-iodine causes 
first a carmine-red color and finally a reddish-brown precipi- 
tate, which gradually bleaches out. 

Nitrates. — When nitrates are present in a solution, a drop 
of barium chloride added to a drop of the solution will produce 
a precipitate of octahedral crystals of barium nitrate. See 
also under Diphenylamine in the last chapter. 

Nucleus. — The nucleus can best be demonstrated in tissues 
which have been fixed according to the directions given under 
Fixatives in the last chapter. Also under Iodine-green and 
Acetic Acid, and Methyl-green and Acetic Acid, are given 
directions for instantly fixing and staining nuclei. The three- 
color method of staining detailed on page 231 gives the best 
results for the dividing nucleus. 

Oils. — Ethereal and fatty oils have already been discussed 
under separate heads, where the methods for distinguishing 
between the two will be found. See also in the last chapter 
under Alcannin, Cyanin, and Osmic Acid. 

Oxalic Acid. — When calcium nitrate is added to sections 
containing oxalic acid, crystals of calcium oxalate are formed. 
With uranyl acetate crystals of uranium oxalate are formed 
in tissues containing oxalic acid. The crystals are rhombic, 
of rectangular form, and when large, appear of a yellow color, 
and, being doubly refractive, they may be studied to advan- 
tage with the polarizer. 

Paragalactan. — This occurs as a thickening of the cell- 
walls in the cotyledons of Lupinus luteus. When it is heated 
with nitric acid, mucic acid is formed, and when heated with 
dilute sulphuric acid, galactose, C 6 H 12 6 , and a pentaglucose 
are formed. When heated with phloroglucin and hydrochloric 
acid, a cherry-red color is produced. Paragalactan is not dis- 



PARAGAL \CTAN — PECTIC COMPOUNDS 

solved by cuprammonia, and is stained slightly or not at all 
by chloroiodide of zinc. 

Paramylum. — Paramylum grains are flattened, cylindrical. 
stratified bodies occurring in the bodies of the Euglenae and in 
the cysts of Leptophrys vorax. The paramylum grains are 
hardly affected by water, alcohol, ether, nitric acid, or concen- 
trated chromic acid; and while they are hardly soluble in 5 
per cent, potassium hydrate, they are easily soluble in a 6 per 
cent, solution. They may also be dissolved in concentrated 
sulphuric acid. They are not stained by iodine, chloroiodide 
of zinc, or by any of the organic coloring matters. 

Pectic Compounds. — The pectic substances (pectin, pec- 
tose, and pectic acids) are widely distributed in the membranes 
of plants. Pectose occurs associated with cellulose in the 
membranes of embryonic tissues, where it is distributed 
throughout the entire thickness of the membrane. Pectose 
also occurs in most lignified, suberized, and cutinized mem- 
branes. The middle portion of cell-walls — the so-called mid- 
dle lamella — consists, for the most part, of calcium pectate. 
When thin sections of plant tissues are treated for several 
hours with a mixture of 1 part of hydrochloric acid and 4 
parts of alcohol, the calcium pectate becomes changed, so that 
pectic acid is liberated and calcium chloride is formed. The 
pectic acid is insoluble in water, but is soluble in a 10 per cent, 
solution of ammonia, so that after rinsing the sections in water 
and treating with the ammonia solution, the cells may be sepa- 
rated from each other by a slight pressure on the coverglass. 
When the sections are placed for a considerable time in cold 
alkaline solutions, a double pectate is formed which swells in 
cold water and finally dissolves in it. After the calcium pec- 
tate of the middle lamella has been removed, the pectose which 
permeates the cell-wall still remains, but by treatment with 
cuprammonia it may be removed from sections which have 
already been acted on by dilute hydrochloric acid. The pectic 
substances may be stained only in neutral or slightly acid solu- 
tions. For this reason it is a good plan to place sections for 



324 MICROCHEMISTRY OF PLANT PRODUCTS 

a short time in a 3 per cent, solution of acetic acid, and then 
to wash them in water before transferring them to the stain- 
ing solutions. Safranin, methylene-blue, bleu de nuit, and 
ruthenium-red are excellent stains for pectic substances. Saf- 
ranin stains the protoplasts and the lignified, suberized, and 
cutinized cell-membranes a cherry-red, while the pectic com- 
pounds are stained orange-yellow. Methylene-blue and bleu 
de nuit stain the protoplasts and the lignified membranes blue, 
and the pectic substances a violet color. See also in the last 
chapter under Ruthenium-red. 

Pezizin. — Pezizin is an orange-red coloring matter which 
occurs in solution within the paraphyses of Peziza aurania and 
P. convexula. It is soluble in alcohol and ether, and is not 
altered by alkalies and organic acids. It dissolves without 
color in hydrochloric acid and is colored bright green by nitric 
acid. 

Phloridzin, C 21 H 24 O 10 . — A glucoside occurring in the 
leaves and in the cortex of the roots and stems of the Pomacese. 
When tissues of Pirus malus containing phloridzin are treated 
with ferric chloride, a dark brown solution is formed, while 
treatment with ferrous sulphate causes a yellowish-brown pre- 
cipitate. The tissues of the pear, cherry, and plum are apt to 
contain large amounts of tannins which produce a green color 
with salts of iron, and so mask the phloridzin reaction. 

Phloroglucin, C 6 H 3 (OH) 3 . — This occurs in solution in the 
cell-sap. To demonstrate its presence treat previously dried 
sections with a solution prepared by dissolving 0.005 §" m - °£ 
vanillin in 0.5 gm. of alcohol, and adding 0.5 gm. of water 
and 3 gm. of concentrated hydrochloric acid. When phloro- 
glucin is present, this solution produces a light red color. 

Phosphoric Acid, H 3 P0 4 . — This can be best demonstrated 
in the ash. The ash is dissolved in hydrochloric acid and the 
solution is evaporated to dryness; then the residue is treated 
with ammonium molybdate, which, if phosphoric acid is pres- 
ent, produces a precipitate of ammonium phospho-molybdate, 
the crystals of which have a greenish-yellow color under the 



PHOSPHORIC ACID PIPERINE 325 

microscope. If the presence of phosphoric acid is to be sought 
for in fresh tissues, sections should be heated in a drop of 
ammonium molybdate on the glass side. This method also 
produces a precipitate of crystals of ammonium phospho- 
molybdate in the presence of phosphoric acid. If ammonium 
tartrate is present in the tissues, ammonium molybdate does 
not serve so well as a test for phosphoric acid. In such a case 
a solution should be used, consisting of 25 volumes of a con- 
centrated aqueous solution of magnesium sulphate, 2 volumes 
of a concentrated aqueous solution of ammonium chloride, and 
1 5 volumes of water. With phosphoric acid this solution pro- 
duces a precipitate of ammonio-magnesium phosphate the crys- 
tals of which are frequently formed in x- and *-shaped clusters. 

Phycoerythrin. — The red coloring matter in the Florideae 
or red algae. It is soluble in fresh water, leaving chlorophyll 
behind in the plastids, while in ether the chlorophyll is ex- 
tracted and the phycoerythrin is left. 

Phycocyanin. — The blue coloring matter in the blue-green 
algae. It is soluble in cold water, glycerine and alkalies, 
giving a blue solution with red fluorescence. It is insoluble in 
alcohol and ether. 

Phycophaein. — The brown coloring matter of the brown 
algae. It is soluble in fresh water and more readily in hot 
water, leaving chlorophyll and carotin behind in the plastids. 
It is insoluble in strong alcohol, ether, etc. 

Piperine, C 17 H 19 N0 3 . — Piperine is an alkaloid occurring 
in the fruit of the Piperaceae, and notably in black pepper, and 
it has not been found outside this family. Very thin sections 
may be rubbed out somewhat under a coverglass to press out 
the ethereal oil, which will then evaporate and leave the piper- 
ine to crystallize in the form of minute short needles. A sec- 
tion becomes of a deep red color when treated with concen- 
trated sulphuric acid, while with nitric acid an orange color 
is produced. When sections are moistened with sodium mol- 
ybdate, and then treated with concentrated sulphuric acid, they 
take on a blue color. Piperine is easily soluble in acetic acid. 



326 MICROCHEMISTRY OF PLANT PRODUCTS 

Proteids (Albuminoid Substances). — Proteids are stained 
from yellow to brown by a dark solution of potassium iodide- 
iodine. The dilute solution of iodine recommended for starch 
should not be used, for proteids are stained less readily than 
starch. Millon's reagent (see under this head in the last chap- 
ter) colors proteids a brick-red color. If the solution is old 
and has lost its efficiency, a few drops of a solution o*f potas- 
sium nitrate will probably restore it. Concentrated nitric acid 
colors proteids yellow, and the addition of ammonium pro- 
duces a still deeper yellow. When sections lie for an hour 
or two in a solution of i gm. of sodium phospho-molybdate 
in 90 gm. of distilled water and 5 gm. of nitric acid, which has 
been filtered after standing for several days, the proteid sub- 
stances appear in the form of yellowish granules. A concen- 
trated solution of nickel sulphate colors proteid granules yel- 
low or blue. When rather thin sections are placed in a con- 
centrated solution of copper sulphate for about half an hour, 
and then are placed in water for about an hour, and then are 
transferred to a concentrated solution of potassium hydrate, 
proteids are colored red or violet, which becomes deeper when 
the solution in which the sections are lying is heated .somewhat. 
The pepsin-glycerine and pancreatin-glycerine ferments pre- 
pared by Dr. G. Griibler in Leipzig are solvents of proteids. 
Sections are treated for an hour at a temperature of 40 C. 
with a mixture of 1 part of. pepsin-glycerine and 3 parts of 
water, to which is added 0.2 per cent, of chemically pure hy- 
drochloric acid. Pancreatin-glycerine is employed in the 
same manner as the pepsin-glycerine. 

Protein Crystalloids. — Under Aleurone in this chapter 
are given methods for differentiating crystalloids in aleurone 
grains. Protein crystalloids also occur in the cytoplasm, nu- 
cleus, and chromatophores, and in all of these cases the crys- 
talloids have essentially the same nature, but they may vary 
considerably in form. For staining crystalloids, see also in 
the last chapter under Acid Fuchsin. 

Protoplasm. — The protoplasm of the cell can be studied to 



PROTOPLASM 327 

advantage by means of the microscope only after being killed 
and fixed by such reagents as those formulated under Fixa- 
tives in the preceding chapter. The different constituents of 
the protoplasm can then be differentiated by means of stains 
or by means of digestive ferments, such as pepsin and pancrea- 
tin. Iron hematoxylin, or a combination of fuchsin and io- 
dine green, or of safranin, gentian violet, and orange G, are 
specially to be recommended for differentiating the different 
parts of the protoplasm. For staining the leucoplasts and 
chromatophores in general, see under Acid Fuchsin, page 268. 
Protoplasmic Connections. — The protoplasmic connec- 
tions between the plates of sieve tubes may be strongly stained 
by acid fuchsin and aniline water (see page 269). More deli- 
cate protoplasmic connections require the use of a swelling 
agent for their demonstration. Sections of fresh material 
may be fixed with a solution of 0.05 gm. of iodine and 0.2 
gm. potassium iodide in 15 gm. of water, and then the iodine 
should be replaced by chloroiodide of zinc, which should be 
allowed to act for about 12 hours. At the end of this time 
the membranes traversed by the protoplasmic connections will 
be swollen to greater or less extent, so that the chloroiodide 
of zinc may be washed out in water and the sections stained 
by acid fuchsin and aniline water, as already suggested. Sul- 
phuric acid may be used instead of the chloroiodide of zinc 
as the swelling agent. For demonstration purposes sections 
through the endosperm of the Gramineae, or tangential sections 
through the green bark of Rhamnus frangula, may be used. 
Sections are placed on a coverglass in a drop of sulphuric acid. 
After a few seconds the acid is washed away by immersing the 
coverglass and moving the sections about in a dish filled with 
water. The sections remain in the water for only a short 
time, and are then to be stained in an aqueous solution of ani- 
line blue, washed in water, and mounted for examination in 
dilute glycerine. Or the sections may be stained iu a satu- 
rated solution of picric acid in 50 per cent, alcohol, to which 
aniline blue is added until the solution has a blue-green color. 



328 MICROCHEMISTRY OF PLANT PRODUCTS 

Pyrenoids. — The pyrenoids may be simultaneously fixed 
and stained by placing the material in a concentrated solution 
of picric acid in 55 per cent, alcohol, to a watch-glass of which 
is added about 5 drops of the acid fuchsin and aniline water 
solution described on page 269. The material should remain 
for about 2 hours in a watch-glass of this solution. It should 
then be washed for a quarter of an hour in alcohol and 
mounted for examination in dilute glycerine. If permanent 
mounts are desired, the material should be placed in a watch- 
glass of dilute glycerine, which should then be allowed to con- 
centrate in a place free from dust. The material should finally 
be mounted in glycerine- jelly. The material may be mounted 
in Canada balsam by transferring it from the alcohol in which 
it was washed to successively stronger solutions of balsam in 
xylol until the ordinary solution used for mounting is reached. 

See under Dahlia in the previous chapter for other methods 
of treating pyrenoids. 

Reserve Cellulose. — Those hemicellulose thickenings of 
cell-walls in seeds, etc., which are essentially reserve food ma- 
terials, and are made soluble by diastatic ferments, and em- 
ployed as food material in the germination of seed, are known 
as reserve cellulose. Sections taken from a sprouted date seed 
and treated with potassium hydrate and stained with alizarine 
show the inner layers of the cell-walls which have been acted 
on by the diastase unstained, while the outer layers which 
have not yet been affected by the diastase are stained an intense 
violet. If Congo-red is used instead of the alizarine, the in- 
tact layers are scarcely stained, while the layers which have 
come under the influence of the diastase are stained a dark red. 
See under Hemicellulose. 

Resin.— When sections containing resin are treated for 
some time with a tincture of alcannin, the resin assumes a red 
color. When sections from tissues which have lain for about 
a week in a concentrated aqueous solution of copper acetate 
are examined under the microscope, the resin will be seen to 
be colored an emerald-green. 



RUBERYTHRIC ACID — SAPONIN 329 

Ruberythric Acid, C 2(> II 2S 14 . — This glucoside occurs in 
the roots of Rubia tinctorium, and is the chief constituent of 
the madder dye obtained from the roots of this plant. It gives 
a yellow color to the cell-sap of the young roots ; the cell-walls 
of old roots, however, have absorbed it and are colored by it. 
It is colored a purple-red by potassium hydrate, and an orange 
color by acids. In dry roots it takes on the form of red flakes, 
and in the injured cells of fresh material it assumes the same 
form. It may be extracted by alcohol from its yellow solution 
in uninjured tissues, but in the red flake form it is not dis- 
solved by alcohol. 

Rutin, C 42 H 50 O 25 . — This glucoside is widely distributed in 
plants. It crystallizes from an aqueous solution in the form 
of minute light yellow crystals. The yellow color of straw is, 
in part, due to it. When treated with ammonia or lime-water, 
rutin forms a deep yellow solution, which turns to brown on 
exposure to the air. 

Salicin, C 13 H 1S 7 . — Salicin is a glucoside which occurs in 
particular abundance in the cortex of many poplars and wil- 
lows. It may be dissolved by water, but more readily by boil- 
ing water, by aqueous solutions of alkalies, and by acetic acid. 
It is insoluble in ether. It crystallizes in the form of needles, 
scales, or thin plates. It is colored by concentrated sulphuric 
acid, and, on the addition of a little water, a red powder is 
thrown down in the sulphuric acid solution. 

Santalin. — Santalin is the coloring matter of the red san- 
dal-wood, Pterocarpus santalinus. It is insoluble in water, 
but is soluble in ether with a yellow color, and with 80 per 
cent, alcohol it gives a blood-red solution. Stronger alcohols 
give the same result. It is also soluble in acetic acid and in 
aqueous alkaline solutions. 

Saponin, C 19 H 30 O 10 . — This glucoside occurs in solution in 
the cell-sap of many Leguminosse. Quillaja saponaria con- 
tains in the bark 2 per cent, of saponin. It is easily soluble in 
water and is precipitated from solution by the addition of 
strong alcohol. When treated with a mixture of equal parts 



33° MICROCHEMISTRY OF PLANT PRODUCTS 

of alcohol and sulphuric acid, a yellow color is produced which 
soon changes to red, and later to violet. If it is then treated 
with a concentrated solution of chloride of iron, a brown or 
bluish-brown precipitate is formed, the intensity of the bluish 
color increasing with the amount of saponin present. 

Seminose. — Seminose is one of the products resulting from 
the hydrolysis of hemicellulose by sulphuric acid. It is dex- 
trorotary, reduces Fehling's solution, and is fermentable. 

Silica, Si0 2 . — Silica occurs in the skeletons of diatoms, and 
as incrustations over the epidermis of the Equisetaceae and 
Graminese. It also sometimes occurs in masses in the interior 
of cells. It may be isolated from the organic matter with 
which it is associated by burning over a flame bits of epider- 
mis incrusted by it, or diatoms, which are placed in a drop of 
concentrated sulphuric acid on a piece of platinum foil. By 
this treatment the organic matter will be destroyed, and the 
silica will remain behind as a pure white ash. The silica may 
also be obtained pure by placing bits of tissues incrusted by it 
in a drop of concentrated sulphuric acid, and then after a time 
adding 20 per cent, chromic acid, and following this with 
additions of still stronger chromic acid until a considerable 
strength has been reached, and, finally, washing in water and 
alcohol. Silica is distinguished by being insoluble in all the 
acids excepting hydrofluoric acid. Silicious skeletons may be 
removed from diatoms by placing the latter in hydrofluoric 
acid which is contained in a platinum vessel. The vessel 
should be kept on a water-bath, and the diatoms should re- 
main in the acid for 24 hours. At the end of this time the 
acid should be thoroughly washed out from the diatoms. On 
examination with the microscope, the diatoms will then be 
found to have lost their silicious skeletons. In some instances 
a thin exterior membrane which is stained brown by iodine is 
to be observed ; but in other instances this membrane has been 
a too insignificant part of the skeleton to retain its identity 
after the removal of the silica. 

Sinapine, C 16 H 32 N0 5 . — This is an alkaloid occurring in 



SINAPINE STRYCHNINE 331 

the seeds of the white mustard. When sections of these seeds 
are placed in a concentrated solution of potassium hydrate, 
they assume a yellow color, which changes to orange on warm- 
ing. This reaction loses some of its value, however, from the 
fact that a glucoside called sinalbine also occurs in the seeds 
of the white mustard and turns yellow on the application of 
potassium hydrate. 

Solanin, C 2S H 47 NO n + H 2 0. — This glucoside occurs in 
the tissues of Solarium tuberosum, in the berries of Solanum 
nigrum and S. Dulcamara, and in many other species of the 
Solanaceae. To demonstrate its presence, sections should be 
placed in a mixture of i part of ammonium vanadate and 1,000 
parts of a mixture of 98 parts of sulphuric acid with 36 parts 
of water. This produces with solanin a yellow color, which 
changes successively into orange, purple-red, brown, red- 
orange, carmine-red, raspberry-red, and blue-violet. The 
color then passes into a grayish-blue and disappears. With 
concentrated sulphuric acid solanin assumes a yellow color, 
which changes to red, and then violet, and then passes into 
gray and disappears. 

Starch, C 6 H 10 O 5 . — A solution of potassium iodide-iodine 
stains starch from pale violet to purple, depending on the 
strength of the iodine solution. Chloroiodide of zinc stains 
starch-grains purple, and at the same time swells them. A solu- 
tion of chloral hydrate and iodine dissolves the protoplasmic 
cell-contents and stains included starch-grains purple. This 
reagent is particularly adapted to demonstrate the presence of 
starch in chloroplasts or amyloplasts. The bleaching effect 
of the chloral hydrate is so great that starch may be demon- 
strated in whole leaves by the chloral hydrate and iodine re- 
agent. For the further treatment of starch with reagents, see 
in the preceding chapter under Eau de Javelle, Calcium Ni- 
trate, Diastase, Methyl-violet, Silver Nitrate. For the struc- 
ture of starch-grains, see page 180. 

Strychnine, C 21 H 22 N 2 2 . — This alkaloid occurs associated 
with brucine in the seeds of Strychnos nux-vomica, S. multi- 



33 2 MICROCHEMISTRY OF PLANT PRODUCTS 

flora, and S. Malaccensis. When sections containing strych- 
nine are treated with a solution of i gm. of ammonium vana- 
date in ioo c.c. of sulphuric acid, they quickly take on a violet- 
red color, which after a time changes to brown. If sections 
of the seeds of Strychnos nux-vomica are treated with sul- 
phuric acid containing an excess of eerie sulphate, the walls 
of the cells are colored a bluish-violet. The sections must 
have been previously treated with petroleum ether and abso- 
lute alcohol to remove the fatty oils, grape-sugar, and brucine. 
The reagent should be applied immediately before the obser- 
vation is to be made. If sections are treated with concen- 
trated sulphuric acid, and crystals of potassium bichromate are 
then added, a violet color is produced. 

Suberin and Suberized Walls. — Suberized walls are 
stained green when treated for about an hour in the dark by 
a freshly-prepared strong solution of chlorophyll. A cold 
concentrated solution of potassium hydrate colors suberized 
walls yellow. When the potassium hydrate is heated yellow 
drops and granular masses are formed. When suberized 
walls are heated in a solution of potassium chlorate in nitric 
acid, they become changed into droplets which melt between 
30 and 40 ° C, and which are soluble in hot chloroform, alco- 
hol, ether, benzol, or dilute potassium hydrate. At ordinary 
temperatures concentrated chromic acid solutions have little 
effect on suberized walls. A solution of potassium iodide- 
iodine, and chloroiodide of zinc colors suberized membranes 
from yellow to brown. After long treatment with a dilute 
solution of potassium hydrate, suberized membranes may be 
stained violet with chloroiodide of zinc. Alcannin stains 
suberized walls red. Under the polariscope suberized walls 
are seen to be doubly refractive. They lose this property on 
heating and regain it on cooling. It may be deduced from 
this that the constituents of the walls are in part, at least, in 
crystals which are melted by heat, but reappear on cooling. 
See under Methyl-blue and Cyanin. 

Syringin. — Syringin is a glucoside occurring in the cortex, 



sVRINGIN TANNINS 333 

and to a certain extent in the xylem and medullary rays of 
Syringa vulgaris, Robinia pseudacacia and species of Ligus- 
tnun. It is especially abundant in early spring. Sections 
containing syringin, when treated with concentrated sulphuric 
acid, acquire a dark blue color, which changes to violet. Nitric 
acid dissolves syringin with a blood-red color. Syringin crys- 
tallizes from aqueous solutions in the form of colorless, needle- 
like crystals which are grouped in the form of a star. The 
crystals are dissolved with difficulty in cold water, but readily 
in boiling water or in alcohol. 

Tannins. — Various substances occurring in plants having 
an astringent taste, and turning dark blue or green with salts 
of iron, are termed tannins or tannic acid. Tannins occur in 
greatest abundance in the bark and in pathological gall forma- 
tions. Oak-galls furnish excellent material for the demon- 
stration of tannin. When sections are treated with an aqueous 
solution of ferric chloride, they take on a deep blue color, due 
to the presence of tannin. Aqueous solutions of ferrous sul- 
phate give the same result. If the reaction is watched under 
the microscope, it is noticed that at first a deep blue precipitate 
is formed, which soon dissolves and imparts its color to the 
surrounding fluid. When sections are placed in a 10 per cent, 
aqueous solution of potassium bichromate, a flocculent reddish- 
brown precipitate is formed in the tannin-bearing cells. When 
sections are placed in a concentrated solution of ammonium 
molybdate in concentrated ammonium chloride, the same char- 
acter of precipitate is produced as when potassium bichromate 
is used. Lead acetate produces a white precipitate with tan- 
nins. The following method may be employed : Sections are 
placed in a 7 per cent, solution of copper acetate for about a 
week or longer, and are then placed on a slide in a drop of 
a 0.5 per cent, solution of ferrous sulphate. After a few min- 
utes, and before the cell-walls begin to turn brown, the sections 
are washed in water and transferred to a watch-glass of alco- 
hol to drive out air-bubbles and extract chlorophyll, if any is 
present. The sections are then mounted in glycerine for 



334 MICROCHEMISTRY OF PLANT PRODUCTS 

examination under the microscope. By this treatment an in- 
soluble brown precipitate is produced in the presence of tan- 
nins. The sections may be transferred from the glycerine to 
glycerine- jelly if permanent mounts are desired. If the sec- 
tions are taken from the alcohol in which they were placed to 
remove the chlorophyll, etc., and placed in a solution of iron 
acetate, a blue or a green color will be produced, according to 
the kind of tannin present. If it is desired to fix the cell- 
contents while testing for tannins, the sections should be placed 
in a concentrated alcoholic solution of iron acetate instead of 
in the aqueous solution, as above. When living tissues are 
placed in a solution of I part of methyl ene-blue in 500 parts 
of distilled water, those cells which contain tannins take on a 
blue color, and later a deep blue precipitate is formed in these 
cells. Cells containing phloroglucin act in the same way to 
this reagent as those containing tannins. 

Theobromine, Dimethyl-xanthin, C 7 H 8 N 4 2 . — This al- 
kaloid occurs in the cocoa-bean and in different parts of several 
species of Theobroma. Its presence may be demonstrated by 
the use of hydrochloric acid and chloride of gold, as directed 
under Caffeine. The reactions for caffeine and theobromine 
are sometimes difficult to distinguish. When sections con- 
taining theobromine are heated in distilled water on the slide 
to the boiling-point, and the sections are allowed to dry slowly, 
and a drop of benzol is added to the residue, crystals of theo- 
bromine appear in the form of a fine powder on the evapora- 
tion of the benzol ; whereas, when sections containing caffeine 
are treated in like manner, the crystals containing caffeine take 
on the form of needles. 

Tyrosin, C 6 H 4 OH.CH 2 .CHNH 2 .COOH.— Tyrosin may be 
demonstrated in abundance in the tubers of the Dahlia. When 
sections are mounted under a coverglass in glycerine for sev- 
eral days, needle-shaped crystals of tyrosin are deposited in 
radiating groups. In an abundance of glycerine the crystals 
are not deposited, for the reason that the tyrosin becomes too 
much diffused through the glycerine. The crystals appear 



TV ROSIN — wax 335 

brownish by transmitted, and white by reflected, light. When 
a portion of a Dahlia tuber is placed in a dish of about the 
same size as itself, and covered for about two thirds of its 
length with alcohol, an abundance of tyrosin crystals will col- 
lect at the exposed cut surface. The crystals of tyrosin are 
colored a deep red by means of Millon's reagent, and when 
nitric acid is poured over them, and then evaporated, a yellow 
residue is left. 

Vanillin, C (; H v OH.OCH,.CHO.— The aldehyde vanillin 
occurs abundantly in the dry, but not in the fresh, pods of 
Vanilla. It is often found in a crystalline condition on the 
surface of dried pods. It is soluble in alcohol and ether, and 
to a certain extent in hot water, but it is soluble with difficulty 
in cold water. When sections containing vanillin are wetted 
with a 4 per cent, solution of orcin, and then treated with con- 
centrated sulphuric acid, they take on a deep carmine-red 
color ; and when they are treated in like manner with a solution 
of phloroglucin in place of the orcin, a brick-red color is pro- 
duced. It seems probable that vanillin is always present in 
lignified walls, judging from the colors which these assume 
with phloroglucin and orcin. 

Veratrine, 0371153X0! t . — The alkaloid veratrine occurs in 
the tissues of Veratrum album. When sections are placed in 
a mixture of 1 drop of concentrated sulphuric acid and 2 drops 
of water on a glass slide, and examined under a microscope, 
it is to be seen that the walls or cell-contents of the cells con- 
taining veratrine assume a yellow color, which soon changes to 
an orange-red, and finally to a dusky violet. 

Wax. — Wax frequently occurs in plants as a crust-like, or 
granular, or rod-like layer over the cuticle. It consists of 
fats and free fatty acids, together with other substances. Wax 
is insoluble in water, but it will melt and form droplets in 
water at ioo° C. It is hardly soluble in cold alcohol, but will 
quickly dissolve in boiling alcohol. When sections containing 
wax are heated in a solution of alcannin in 50 per cent, alco- 
hol, the wax runs together in droplets, which become stained 



33^ MICROCHEMISTRY OF PLANT PRODUCTS 

red by the alcannin. Wax is not wetted by water, and sec- 
tions are best mounted for study in cold alcohol, which will 
dissolve the wax but little, if at all. 

Wound Gum. — The wounded surfaces of deciduous trees 
become protected by the formation of wound gum from starch 
contained in the live cells. Sections taken through the wounded 
surfaces of such plants several days after the wound has been 
inflicted show brownish granules of wound gum in the medul- 
lary rays, tracheal tubes and wood-cells. The wound gum 
may be found lying free in the cytoplasm, or surrounding 
starch-grains which have contributed to the formation of the 
gum. Wound gum is not soluble in warm water, but may be 
dissolved in hot nitric acid or in eau de Javelle after several 
hours. It is not soluble in sulphuric acid, potassium hydrate, 
alcohol or ether, but it may be dissolved in alcohol after treat- 
ment for a few minutes with a solution of potassium chlorate 
in dilute hydrochloric acid. It may be stained with a solution 
of fuchsin, iodine green, safranin, or methyl-green. It is 
stained red by phloroglucin and hydrochloric acid. 

Xanthine, C 5 H 4 N 4 2 . — Xanthine occurs in an amorphous 
condition or in the form of granules in yellow chromoplasts. 
It differs from carotin in being soluble in alcohol, and in being 
deposited in amorphous and resin-like masses on the evapora- 
tion of its solvent. It is but little soluble in ether and benzine. 
Some varieties of xanthine are soluble in water while others 
are not. It becomes green and then blue when treated with 
sulphuric acid, and with potassium iodide-iodine it is colored 
green. 



CHAPTER XVII 
DETECTION OF ADULTERATIONS IN FOODS AND DRUGS 

How to Begin. — The microscope is indispensable to the 
detection of adulterations in powdered foods and drugs, and 
the enforcement of our pure food laws will require men skilled 
in the application of the microscope to this kind of research. 

Before proceeding with the microscopic examination of a 
powder a knowledge is necessary of the histology of the plant 
part which is supposed to constitute the powder. If, for ex- 
ample, powdered cinnamon is to be investigated the different 
kinds of cinnamon barks on the market must be obtained for 
study, from a reliable source, and these must be examined in 
cross and longitudinal sections and in powdered form. The 
directions that will now be given for the study of cinnamon 
bark will serve for dried barks and woods in general. The 
bark is hard and brittle and will need to be put in better condi- 
tion for sectioning. Place it in warm water and let it soak 
over night, and then transfer it to equal parts of alcohol, 
glycerine and water and let it remain there for two weeks — 
the longer the better. It can then be sectioned free-hand (see 
page 218), while held between two pieces of cork free from 
grit; or it can be sectioned in a sliding microtome (Fig. 127), 
Keep the knife wet with 70 per cent, alcohol and transfer the 
sections to a watch-glass of water. Mount the sections for 
the first study in a drop of dilute glycerine. Water would do 
but it may evaporate before the examination is completed. If 
it is found that the thinnest sections are too opaque to make 
the cells out clearly they may be remounted in a drop of satu- 
rated chloral hydrate solution. In this case or in any other 
where the material has become dry and brown if the chloral 
hydrate does not clear the sections well enough they may be 
23 337 



33$ DETECTION OF ADULTERATIONS 

left over night in hydrochloric acid 10 parts and water 90 
parts and then remounted in chloral hydrate. This clears up 
very refractory subjects. 

When the sections are in condition to show all of the cells 
clearly make camera lucida drawings (see page 247) from both 
cross and longitudinal sections showing groups of cells from 
each tissue to compare with similar drawings from the powder 
under investigation. This method of comparison is much 
more reliable than one in which the drawings are not made. 
If the full length of the bast fibers cannot be seen in the 
sections the fibers can be isolated by the methods for macera- 
tion given in Chapter XV under Maceration. 

Employment of Micro-chemistry. — Having thus become 
acquainted with the different tissues mount a section in chloral 
hydrate iodine (see page 259) to bring out clearly any starch 
that may be present. This reagent dissolves proteids and 
swells the starch-grains and colors them blue so that they can 
easily be seen even when very minute and previously obscured 
by the cell contents. Drawings had better be made of the 
starch-grains, but since they are swollen in the chloral hydrate 
other sections mounted in potassium iodide-iodine (see page 
278) should be used for the drawings. Other sections should 
be tested for aleurone (page 298), gums (page 316), mucilage 
(page 320), resins (page 328), and tannins (page 333). 

The nature of the cell-walls is next to be tested. Mount 
sections in chloroiodide of zinc (page 259), and stone cells, bast 
fibers and cork cells should be colored yellow; and the walls of 
other tissues should be purple, indicating cellulose. Mount 
sections in phloroglucin (page 292), and bast fibers and stone 
cells only will be colored pink; while other sections mounted 
in aniline sulphate (page 254) will have yellow bast fibers and 
stone cells and all other tissues unstained. Phloroglucin and 
aniline sulphate, since they stain only lignified walls, are of 
especial value in sharply differentiating stone cells, bast fibers 
and wood fibers and the tracheal tissues from all other tissues. 

Finally the sections are to be studied with a polariscope 



H'AKI.SOX OF AUTHENTIC AND SUSPECTED TOWDERS 339 

I page 250) to bring out minute crystals that would otherwise 
escape detection. 

Having studied sections of cinnamon bark in this manner 
one is prepared to recognize the different tissues in the state 
of powder. Grind the bark in a perfectly clean mill or with 
a pestle and pass it by shaking, and not pressure, through the 
series of fine sieves 20, 40, 50, 60, 80, of the U. S. Pharma- 
copoeia. The bark should be ground to the degree of fine- 
ness that results in the same amount of residuum on each sieve 
as results from an equal weight of the powder whose purity 
is under investigation. Any fragments too large to pass 
through sieve 50 will need further pulverization before their 
cell elements can well be made out under the microscope. 

The authentic and the questionable powders are now to be 
compared under the microscope. Take equal amounts of each 
powder of the same degree of fineness and shake them up in 
equal small quantities of water 2 parts and glycerine 1 part; 
and while still in agitation mount a drop of each of these mix- 
tures under coverglasses of equal sizes. The object is to 
compare the two powders under as like conditions as possible. 

From both preparations make camera lucida drawings of 
the different cells and cell-contents as they lie in the field, so 
that by a comparison of the sizes, shapes, frequencies, etc., of 
the elements of the two preparations it may be determined 
whether foreign substances are present in the powder under 
investigation. 

If it proves that the tissue fragments of the powders are too 
opaque to allow the shapes and sizes of the cells and the thick- 
ness and markings of the walls to be made out with certainty 
the pow r ders should be first bleached by the hydrochloric acid 
and chloral hydrate treatment recommended above for the 
sections. 

By the above method, while the forms of the cells and the 
characteristics of their walls are plainly revealed, there may be 
certain cell-contents, such as some classes of proteids, that 
will have gone into solution, and the powders should be further 



34° DETECTION OF ADULTERATIONS 

compared by stirring them up and mounting them in a mix- 
ture of castor oil I part and 95 per cent, alcohol 2 parts that 
has been slightly colored with eosin. This will preserve the 
form of proteid cell-contents and stain them pink. 

Various adulterants have been detected in ground cinnamon : 
wood and leaves from the cinnamon tree, sawdust from dark- 
colored woods, ground nutshells and foreign barks, oil cakes, 
various cereal products browned to the color of cinnamon, such 
as bread or biscuit and ground millet. The presence of cereals 
will be apparent from the foreign starch, and nutshells will be 
revealed by the large percentage of stone cells ; and any adul- 
terant will cause a marked difference in the camera lucida 
drawings of the two preparations. 

Sometimes ground cinnamon contains cinnamon bark from 
which the essential oil has been extracted, and then the powder 
may have the same appearance under the microscope as that 
made from the unextracted bark, except that the starch-grains 
will have become swollen and broken in the process of dis- 
tillation. 

If it is found desirable the powders may be treated with any 
or all of the reagents recommended for cinnamon bark, and 
in doing so it would be advisable to make a fresh mount di- 
rectly into each reagent employed. 

Determining the Source of the Adulterant. — Of course 
the possible sources of adulteration are innumerable, but it 
may be taken for granted that those things will be chosen 
which are the cheapest and most available, and which at the 
same time afford the least opportunity for detection. Of all 
adulterants the hardest to detect are those which have no well- 
pronounced anatomical characteristics, or those that can be 
considered adulterants only because some of their useful sub- 
stances have been extracted from them, as when ground cin- 
namon from which the oil has been extracted is mixed with 
the ground unextracted bark. And adulterants easiest to 
detect are those that contain an abundance of characteristic 
starch-grains, such as that from potato and corn, or a large 



COFFEE SPICES 34 • 

amount oi stone cells, as when cocoanut and other nut shells 
are employed. 

Kinds of Adulterants Commonly Employed. — In seeking 
out the fact and source of adulteration under the microscope 
it will be of great assistance to know what kinds of adulter- 
ants have already been found most commonly in use. A list 
of these will now be given. 

Adulterants of Ground Coffee. — Roasted rye, barley, and 
barley malt, ground peas, beans and other legumes, pea hulls, 
and cereals made into pulp with molasses ; ground grape seeds, 
dried, roasted, and ground figs, ground date stones, and cof- 
fee already used in making coffee extract. 

Adulterants of Ground Spices. — The list of adulterants 
here is a long one. Both inorganic and organic substances 
are employed. The inorganic adulterants are : brick dust, 
coal ashes, calcium sulphate and carbonate, sand and clay, 
chrome yellow, and Venetian red. And the organic adulter- 
ants are : hulls and bran of buckwheat, bran and chaff of cere- 
als, flour, mill screenings, peas, beans, and other legumes, cot- 
tonseed and linseed meal, ground cocoanut cake, and other oil 
cakes, ground shells of the cocoanut, almond, and other nuts, 
ground olive stones, sawdust of red sandalwood and of other 
woods, and dyestuffs. 

Black pepper has been adulterated more than any other 
spice. In it has been found almost any variety of w T aste mate- 
rial capable of being reduced to powder. Sometimes when 
the amount of adulteration has been so great as to remove the 
natural pungency very perceptibly cayenne pepper has been 
added. 

The study of adulteration in spices should be undertaken as 
suggested above for ground cinnamon. When the source of 
the spice is in seeds, as in the case of mustard and black pep- 
per, sections of seeds can be obtained by soaking the seeds in 
water and embedding them in glycerine gum, as described on 
page 273. The seeds can then be sectioned free-hand or in a 
sliding microtome. 



34 2 DETECTION OF ADULTERATIONS 

Adulterants of Wheat Flour. — Corn flour and gypsum. 
The starch of the corn flour is quickly detected by the angular 
shape of the grains and the central cracks which are seen with 
especial clearness in alcohol. For the detection of gypsum 
see under Calcium Sulphate on page 306. The presence of an 
inorganic adulterant would be apparent in flour treated with 
a solution of iodine where the starch and proteids would be 
stained and the inorganic matter would be left uncolored. 

Adulterants of Buckwheat Flour. — Wheat flour and corn 
flour. The characteristic starch-grains in each would reveal 
the presence of the adulterant. 

Adulterants of Jams, Marmalades, and Preserves. — Pulp 
of turnip, beet, apple, figs, pumpkin and watermelon ; starch 
paste, gelatine, agar-agar, grass seeds to imitate seed's of ber- 
ries. Adulterants in this class are sometimes very hard to 
detect, partly because of the materials having been macerated 
by cooking, and partly because of the scarcity of characteristic 
elements. Pure fruits raw and cooked, of which the jams, 
etc., are claimed to be made, should be studied under the 
microscope, and the probable adulterants should be studied in 
the same way. Employ aniline sulphate and phloroglucin to 
bring out lignified walls. Dilute some of the suspected prod- 
uct with water, allow it to settle and study the residue under 
high powers, when the presence of silicious diatom skeletons 
would indicate the presence of the seaweed agar-agar. 

Adulterants of Canned Tomatoes. — Pulp of carrots, tur- 
nip, sugar beet, coal tar dyes. 

Adulterants of Sweet Chocolate. — Wheat flour, corn- 
starch, peanut meal, peas, acorns, arrowroot, and cocoa shells. 

Adulterants of Tea. — Tea is sometimes colored with Prus- 
sian blue, indigo, turmeric, soapstone and gypsum. Black tea 
is sometimes coated with plumbago. Under the microscope 
plumbago can be made out by its shining, glossy appearance, 
Prussian blue by its transparent, light blue particles, and in- 
digO' by its greenish-blue particles. The color of Prussian 
blue is removed by sodium hydroxide while that of indigo is 
not. 



PERCENTAGE OF ADULTERATION 343 

The leaves of the following plants have been used as tea 
adulterants : Lithospermum officinale, Epilobium angusti fo- 
lium, E. hirsutum, Salix sp., Fraxinus sp., Sorbus aucuparia, 
Morus alba and nigra, CofTea Arabica, Camellia Japonica, 
Prunus spinosa and avium, Rosa canina, Fragaria vesca, 
Spiraea ulmaria, Wistaria Sinensis. 

It will be seen that in most of these cases the fact of adul- 
teration would be easily detected by the skilled microscopist, 
but the source of adulteration could not be told without a pre- 
liminary knowledge of the histology of the adulterant, and a 
knowledge more intimate than could be obtained from pic- 
tures and descriptions. It is clearly necessary for the investi- 
gator to get a first-hand acquaintance with the possible adul- 
terants. 

Estimating the Percentage of Adulteration. — In some 
instances it is possible to make a very close approximation of 
the percentage of adulteration. Having determined the fact 
and the source of adulteration, make preparation of different 
and definite percentages of adulteration and compare them 
with the sample under investigation for the relative frequen- 
cies of some of the characteristic elements of the adulterant, 
such as starch-grains, bast fibers, stone cells, hairs, etc. If 
these elements are few the total number in the field of view 
may be counted; but if their frequency makes this impossible 
the count may be made in a definite portion of the eyepiece mi- 
crometer. It is very important in doing this that the material 
in all cases be evenly distributed under the coverglass, and 
that a medium power objective be used, in fact as low a power 
as will serve in identifying the different elements, in order that 
the reliability of the results may be increased by a comparison 
of the larger areas which the lower powers embrace. An 
equal distribution of the material under the coverglass can be 
approximated by giving the coverglass a gyrating motion 
under gentle pressure of the forefinger covered with a clean 
cloth. 



INDEX 



Absorption, 93 

Of water and solutes, 97-102 
Aerophytes, 99. 101 
Albumoses, 186 
Alcannin, 252 
Alcohol, 253 
Aleurone grains, 185 
Amides, 186 
Amygdalin, 184 
Amyloid, 183 
Anastomoses, use of, 172 
Aniline oil, 254 

sulphate, 254 
Anthocyanin, 10 
Asparagin, 186 
Assimilation, 196 

Balsam, 254 

Bast fibers, 34, 36, 75, 80, 81, 87 

Benzol, 254 

Bicollateral bundle, 44 

Bleaching, 257, 263 

Bordered pits, no, III, 112 

Borke, 57, 75 

Cambium, 25, 42, 174 
Camera lucida, 247 
Carbohydrate, relation of to nitro^ 

enous foods, 157 
Carbon dioxide, 126, 152 
Carotin, 10 
Carbolic acid, 257 
Cell, definition of, 1 

differentiation, 15 

discovery of, 1 

division, 10 

sap, 3 

wall, 16, 17 
Celloidin, imbedding in, 235 
Cellulose, 259 

cell wall, 16 

reserve. 182 



Chloral hydrate, 259 

Chloroform, 259 

Chloroiodide of zinc, 259 

Chlorophyll solution, 259 

Chloroplasts, 9, 143, 144 

Chromic acid, 260 

Chromoplasts, 9 

Chromosomes, 10 

Circulatory tissues, need of, 160 

Clearing media, 260; carbolic acid, 
257; cedar oil, 258; chloral car- 
mine, 258; chloral hydrate, 259; 
chloral hydrate iodine, 259 ; clove 
oil, 261 ; eau de Javelle, 265 ; 
potassium alcohol, 293; salicylate 
of soda, 294 

Clostridium Pasteurianum, 157 

Clove oil, 261 

Collateral bundle, 44 

Collenchyma, 33, 75, 79, 87 

Collodion, 261 

Companion cells, 37 

Concentric bundle, 44 

Cork, 72, 72, 

cambium, 28, 56 

Corn leaf, cross-section of, 151 

Crude sap, 96 

Cryptogams, vascular, phloem of, 
162 

Cuscuta, 157 

Cuticle, 30 

Cutinization, 66, 68 

Cutinized cell wall, 16 

Cutting sections free-hand, 218 

Cystolith, 211 

Cytase, 195 

Cytological methods, 225 

Cytoplasm, 2, 5, 6 

Dehydrating reagents : Alcohol, 
253 ; aniline oil, 254 



345 



346 



INDEX 



Desert plants absorption of water 

by, 101 
Dextrose, 181 
Diastase, 195, 264 
Diffusion, 97 
Digestion, 194-196, 265 
Dionaea, 196 
Diosmosis, 137 
Diphenylamine, 265 
Drawing board, 248 
Drosera, 196 

Eau de Javelle, 265 

Elaborated sap, 97 

Emulsin, 195 

Endodermis, 25, 36 

Energy, 141, 142, 145; release of, 
126; for food construction, 158; 
supply of, 198 

Engelmann's method, 145 

Enzymes, 195 

Enzyme-secreting cells, 209-311 

Eosin, 266 

Epidermal outgrowths, 70 

Epidermis, 25, 29, 65, 67, 69; mul- 
tiple, 27, 71 

Excretion, nature of, 203 

Euglena, 19 

Euphorbia splendens, 199 

Fascicular cambium, 29 

Fats, 181-182 

Fatty oils, 181 

Fehling's solution, 266 

Ferments, 195 

Fiber-tracheids, no 

Fixatives : Chromic acid, 260 ; cor- 
rosive sublimate, 262; lactic 
acid, 279 ; osmic acid, 291 ; 
picric acid, 292 
and fixation, 267 

Fixing, 226 

Free-cell formation, 13 

Fundamental meristem, 25 

Food, character of circulating, 175 ; 
course of through the stem, 



166-173; digestion of, 194- 
196; kinds of stored, 179; 
nitrogenous, 157; passage of 
from leaf, 167; sources and 
uses of, 141-143; storage of, 
187-191 ; synthesis of with- 
out light, 156; uses of, 197 

Food-building apparatus, 143 

circulation, propelling power in, 
176 

Food-conducting tissues, annual ad- 
ditions to, 173 
storage tissues, extent of, 191- 
194 

Gases, movements, 137-138 

Gentian violet, 270 

Glands, 204 

Gliadin, 186 

Globulins, 186 

Glycerine, 271 ; mounting in, 236 

gelatine, 272 

gum, 273 

-iodine, 273 

jelly, 236 
Glucosides, 184 
Glutamin, 186 
Glutenin, 186 
Ground meristem, 25, 28 
Growth, 21 

in thickness, 48, 56; unusual, 59 
of roots, 55 
Gymnosperms, phloem of, 162 

Hematoxylin, 274 

and safranin, 275 
Hanging drop culture, 275 
Hardening, 227 

processes, 275 
Hydatodes, 213-215 
Hydrogen peroxide, 276 
Humic acid, 98 

Illuminating the microscope, 239 
Imbedding in celloidin, 234 

media : Collodion or celloidin, 
261 ; paraffin, 291 



INDEX 



347 



Infiltration, i~~ 

Intercellular spaces, 126.. 127, 134- 

Interfascicular cambium, 29 
Invertase, 195 
[nulase, 195 
Inulin, 182 

Iodine, 277 

and alcohol, 278 

and glycerine, 278 

green, 279 

and potassium iodide, 278 
Irritability, 20 

Knife, section, care of, 222 
Karyokinetic division, 14 

Laticiferous vessels, 207 

Laevulose, 181 

Leaf, anatomy of, 148; cellular 

architecture of, 150-15 1 ; 

food-building processes in. 

149; vascular bundles in, 166, 

167 
trace, 107 
Leaves, course of food from, 167; 
relation to rings of growth, 115- 
119 
Lenticel, 57, 133-134 
Leucin, 186 
Leucoplasts, 9 
Lignified cell wall, 16 
Lignin, tests for, 254 
Lipases, 195 

Maceration, 279 

Maltase, 195 

Magnification, determination of, 249 

Marchantia, 154 

Medulla, 25 ; ontogeny of, 43 

Medullary rays, 113; frequency of, 
166; functions of, 43, 161, 165, 
166; as storage tissues, 192 

Meristem, 25 

Mesembryanthemum, 199 

Mesophyll, 148 



M icrometer eyepieo 

Microscope, part- of, 238; use of, 
238-251 

Microtome, 220 

Milk tubes, 207 

Millon's reagent, 282 

Minerals, secretion and excretion 

of, 211 
Mitotic division, 14 
Monocotyledons, distribution of tis- 
sues in, 29; growth in thickness 
of, 58; phloem of, 162 
Moss, chloroplasts in, 157 
Mounting media : Boracic acid, 255 ; 
Canada balsam, 230; Vene- 
tian turpentine, 296; glycerine, 
272 ; glycerine gelatine, 272 
sections, 230 
Mucilage, 184 

use of in water storage, 199 
Multiple epidermis, 27, 71 
Myorisin, 195 
Myxomycete plasmodia, 18 

Nepenthes, 196 

Nitella, 17 

Nitrogenous foods, 157 

Nucleins, 186 

Nucleus, 6; division of, 11; powers 

of, 8 
Nutrient media, 282 

Oak wood, 83 
Oleo-resins, 203 
Osmosis, 97 
Oxygen, necessity of, 126 

Palisade parenchyma, 148 
Paraffin, imbedding in, 228 
Parenchyma, 33 

sheath, 45 
Pectase, 195 

Pericycle, 25, 36; in roots, 37 
Periderm, 56 
Phelloderm, 56 
Phellogen, 28; ontogeny of, 56 



348 



INDEX 



Phenol, 257 

Phloem, 25 ; additions to in spring, 
174; constitution of, 37; as 
food carrier, 162; length of 
life of, 174; in Monocotyle- 
dons, 39 ; in Pteridophytes, 
39; relation of to other tis- 
sues, 165 ; relation of one 
year's to that of next, 174 
parenchyma, 37 ; function of, 161 
Phloroglucin, 291 

Photosynthesis, 144, 147; conditions 
affecting, 151; in lower plants, 
153; time required for, 152 
Photosynthetic unit, 146 
Pine wood, in 
Pith, 25 ; ontogeny of, 43 
Plasma membrane, 2, 3, 96; selec- 
tive action of, 98 
Plastids, 9 
Pleurococcus, 154 
Polariscope, 250 
Polytrichum, 155 

commune, 160 
Primary cortex, 25, 31 

medullary rays, 25 ; ontogeny of, 

43 

permanent tissues, 28 

vascular bundle, 37 
Primordial meristem, 25, 27 
Procambium, 25, 27 
Proteids, 184; conduction of, i6t • 
Proteolytic enzymes, 195 
Protoderm, 25, 27 
Protoplasm, 1 

Protoplast, 1, 2 ; chemical and physi- 
cal nature of, 17; powers of, 3, 17 

Radial bundle, 44 
Reagents, use of, 250 
and processes, 252 
Reserve cellulose, 182 
Resin, 203 
Respiration, 126 
Ring of growth, 53, 113-119 



Root hairs, 94, 95, 97; excretions 

from, 96 
Roots, 93 ; growth in thickness of, 

55 
Rubber leaf, cross-section of, 151 

Saccharose, 181 

Sap, crude, 96; elaborated, 97 

Sclerenchyma, 33, 75 

Secondary cortex, 57 

permanent tissues, 29 

Secretion, process of, 212; nature 
of, 203 

Secreting cells and glands, 204-207 

Section-cutting, with microtome, 
220 

Sectioning paraftm material, 229 

Sections, different planes in cut- 
ting, 220 

Section-cutting, free-hand, 218 

Sieve parenchyma, 37 ; ontogeny of, 
38 
tubes, 37 ; chemical analysis of 
contents of, 162; contents of, 
38; duration of functions of, 
175 ; formation of, 161 ; on- 
togeny of, 37 

Skeleton, 78; location of, 88, 89; 
making of, 79; topography of, 86 

Solutes, distribution of, 1 16-120; 
path of from soil, 96 

Sphagnum, 155 

Spirogyra, 154; cell-division in, 14 

Spongy parenchyma, 148 

Stains : Alcannin, 252 ; aniline sul- 
phate, 252; Berlin blue, 255; Bis- 
marck brown, 255; borax car- 
mine, 255; Bordeaux red 256; 
canarin, 257; carmalum, 258; 
chloral carmine, 258; chloroiodide 
of zinc, 259; chlorophyll solution, 
259 ; Congo red, 261 ; corallin, 
261 ; cyanin, 262 ; dahlia, 262 ; di- 
phenylamine, 265 ; eosin, 266 ; 
fuchsin, 268; fuchsin, acid, 268; 
gentian violet, 270; hsematoxylin, 



INDKX 



J49 



274; iodine, 278; iodine green, 
279; methyl blue, 280; methylene 

blue, 280; methyl violet, 282; 

phloroglucin, 291 ; picric acid, 

292; ruthenium red, 294; safranin, 

204 ; silver nitrate, 295 
Staining, 231, 235 
Starch. 179-181 

-heath, 25, 35 
Steapsins, 195 
Stele, 25 
Stomata, 127-133; action of, 130; 

development of, 128 
Stone cells, 33, 75, 84, 85 
Stone-cell tissue, cutting sections of, 

219 
Storage of food, process of, 187- 
191 
of water, 198-200 
Suberized cell wall, 16 
Sunlight, 145 

Tannin cells, 209 

Tillandsia, 101 

Tissue, 25 

Thin-walled parenchyma, 33 

Tracheal elements, 114 

tubes, 39, 105; course of, 106- 

108; as food carriers, 163; 

functions of, 40 ; ontogeny of, 

39 
Tracheids, 39, 109; function of, 40: 

ontogeny of, 40 
Tradescantia, 17; zebrina, 4 
Tubercle bacilli, 157 



Turn-table, 236 
Turpentine, 203 
Tyrosin, 186 

Ulothrix, 20 

Vacuole, 3, 5 

Vascular bundle, 25; growth of, 48; 
types of, 45 
cryptogams, phloem of, 162 
Velamen, 99, 100 

Water, ascent of, 119; devices for 

absorbing, 99-102; distribution of, 

1 16-120; excretion of, 212; path 

of rise of, 127; path of from soil, 

96; in photosynthesis, 153; in 

soil, 94; storage of, 198-200 

Water-conducting tissues, influence 

of environment on, 122 

storage tissues, characteristics 

of, 200 
tubes, 39, 105 
Wood fibers, 39, 82; ontogeny of, 
41 

Xanthin, 10 

Xylem, 25, 87; additions to, 174; 

constitution of, 39; in angio- 

sperms, 39; in gymnosperms. 

50, 109 
parenchyma, 39; functions of, 

40 ; ontogeny of, 40 
Xylene, 279 



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